Science of the Total Environment 408 (2010) 5362–5371
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The chronic toxicity of molybdate to freshwater organisms. I. Generating reliableeffects data
K.A.C. De Schamphelaere a,⁎, W. Stubblefield b, P. Rodriguez c, K. Vleminckx d, C.R. Janssen a
a Laboratory of Environmental Toxicology and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University, Belgiumb Oregon State University, Department of Environmental and Molecular Toxicology, 421 Weniger Hall, Corvallis, OR 97331, USAc Centro de Investigación Minera y Metalúrgica (CIMM), Santiago, Chiled Department for Molecular Biomedical Research, Ghent University, Belgium
⁎ Corresponding author.E-mail address: [email protected] (K
0048-9697/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.scitotenv.2010.07.041
a b s t r a c t
a r t i c l e i n f oArticle history:Received 18 March 2010Received in revised form 24 June 2010Accepted 17 July 2010Available online 1 September 2010
Keywords:Sodium molybdateAquatic toxicitySpecies sensitivity variation
The European Union regulation on Registration, Evaluation, Authorization and Restriction of Chemicalsubstances (REACH) (EC, 2006) requires the characterization of the chronic toxicity of many chemicals in theaquatic environment, including molybdate (MoO4
2−). Our literature review on the ecotoxicity of molybdaterevealed that a limited amount of reliable chronic no observed effect concentrations (NOECs) for thederivation of a predicted no-effect concentration (PNEC) existed. This paper presents the results of additionalecotoxicity experiments that were conducted in order to fulfill the requirements for the derivation of a PNECby means of the scientifically most robust species sensitivity distribution (SSD) approach (also called thestatistical extrapolation approach). Ten test species were chronically exposed to molybdate (added assodium molybdate dihydrate, Na2MoO4·2H2O) according to internationally accepted standard testingguidelines or equivalent. The 10% effective concentrations (EC10, expressed as measured dissolvedmolybdenum) for the most sensitive endpoint per species were 62.8–105.6 (mg Mo)/L for Daphnia magna(21 day-reproduction), 78.2 (mg Mo)/L for Ceriodaphnia dubia (7 day-reproduction), 61.2–366.2 (mg Mo)/Lfor the green alga Pseudokirchneriella subcapitata (72 h-growth rate), 193.6 (mg Mo)/L for the rotiferBrachionus calyciflorus (48 h-population growth rate), 121.4 (mg Mo)/L for the midge Chironomus riparius(14 day-growth), 211.3 (mg Mo)/L for the snail Lymnaea stagnalis (28 day-growth rate), 115.9 (mg Mo)/L forthe frog Xenopus laevis (4 day-larval development), 241.5 (mg Mo)/L for the higher plant Lemna minor(7 day-growth rate), 39.3 (mg Mo)/L for the fathead minnow Pimephales promelas (34 day-dry weight/biomass), and 43.2 (mg Mo)/L for the rainbow trout Oncorhynchus mykiss (78 day-biomass). These effectconcentrations are in line with the few reliable data currently available in the open literature. The datapresented in this study can serve as a basis for the derivation of a PNECaquatic that can be used for nationaland international regulatory purposes and for setting water quality criteria. Using all reliable data that arecurrently available, a HC5,50% (median hazardous concentration affecting 5% of the species) of 38.2 (mg Mo)/Lwas derived with the statistical extrapolation approach.
.A.C. De Schamphelaere).
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© 2010 Elsevier B.V. All rights reserved.
1. Introduction
According to EU Regulation No 1907/2006 concerning theRegistration, Evaluation, Authorization and Restriction of Chemicalsubstances (REACH) (EC, 2006), the registration dossier for high-volume compounds should comply with the data requirements thatare outlined in Annexes VII–X of the REACH legislation. In practice, theminimum requirements include the derivation of a PNECaquaticbased on the lowest chronic toxicity data point of at least three trophiclevels (algae, invertebrates, and fish) onwhich an assessment factor of10 is applied. However, when sufficient data are available, the ECHA
(European Chemicals Agency) guidance on PNEC-derivation allowsthe derivation of a PNEC by means of the scientifically more robuststatistical extrapolation method (ECHA, 2008). This method is basedon a species sensitivity distribution (SSD) which can be developedwhen at least 10 chronic data points representing at least 8 differenttropic levels are available.
An aquatic research project with molybdate as test substance ispresented in this study, and followed the approach that would result inthe most robust PNECaquatic, namely the SSD approach. This researchprogramwas initiated in 2007 by the REACHMolybdenum-Consortiumas part of the REACH dossier preparation, but was taken over by theInternationalMolybdenumAssociation (IMOA) as the data proved to beuseful for broader global regulatory purposes. As a first step, IMOAcommissioned a thorough evaluation of all existing chronic toxicitydata for molybdate in the aquatic environment. Based on the outcome
1 It should be noted that reliable data were available in the literature for some of thesetest species. The rationale for repeating these tests is two-fold: a) Literature data onlybecame available after the testing program was initiated (e.g., data presented in GEI(2009 — data first evaluated in a draft publication by Canton et al., 2006), and b) Largevariation among effect concentrations for single species were noted (e.g., the rainbowtrout O. mykiss); tests were repeated in an effort to confirm previously reported results.
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of this review a testing program was initiated, aimed at generating thedata necessary to 1) develop a species sensitivity distribution formolybdate, and 2) derive a HC5,50% (Hazardous Concentration thataffects 5% of the population) which serves as a reference value forsetting water quality criteria. Results of both the literature review, theecotoxicological testing program, and HC5,50% derivation are presentedin this study.
2. Materials and methods
2.1. Literature review
Studies provided by industry and relevant publications that wereidentified in open literaturewere collected and evaluated according tothe criteria as outlined by Klimisch et al. (1997). In short, thisevaluation procedure allows an objective classification of toxicity datain four different categories (Klimisch 1 to 4), whereby only dataplaced in Category 1 (reliable without restrictions) and Category 2(reliable with restrictions) can be used for risk assessment purposesas stipulated in the REACH legislation. Data that are categorized asKlimisch 3 (not reliable) or Klimisch 4 (not assignable to any of theprevious categories due to lack of information), are considered notuseful.
This Klimisch-scoring takes both the relevance and reliability ofthe reported test results into account. The term relevance refers to thebiological and ecological relevance, relevance of the test substanceand test medium, and relevance of the exposure period. Reliability, onthe other hand, focuses on parameters like adequate description of thetest method and analyses, information on test acceptability criteria,and presence of an effect–concentration relationship.
2.2. Selection of test substance — sodium molybdate
From an environmental point of view the significant Mo(VI) speciesis the simple [MoO4]2− ion since this is the species which enters the cellin plants and animals (Stiefel, 2002; Paue and Lawson, 2002). Thespeciation of molybdenum(VI) in aqueous media as a function of pHand molybdenum concentration has been thoroughly investigated.Under physiological conditions (pHN6.5) the sole molybdenum(VI)species is the molybdate anion, [MoO4]2− (Cruywagen, 2000; Cruywa-gen et al., 2002). Accordingly sodium molybdate, Na2MoO4·2H2O, wasused as source of molybdenum(VI) in the test designs. Othermolybdenum(VI) compounds, e.g. molybdenum trioxide, polymolyb-dates, revert rapidly to the [MoO4]2− ion under environmentallyrelevant test conditions (Greenwood and Earnshaw, 1987).
Some literature data on acute toxicity of molybdenum seemed toindicate that molybdenum trioxide was more toxic compared to othermolybdenum substances (Bentley, 1975). The observed highertoxicity, however, was related to low pH levels (pHb5.5), resultingfrom the transformation of molybdenum trioxide to the molybdate-form. Hence, reported ecotoxicity data did not only reflect molybdatetoxicity, but also reflected pH-toxicity.
2.3. Selection of test organisms
The London Workshop (EC Report, 2001) defined 8 differenttaxonomic groups, listed below, that should be included in the effectsdatabase when the SSD-approach is considered, and this guidancewas adopted in RIP 3.2, Chapter R10 (ECHA, 2008):
• Fish (usually tested species such as salmon, bluegill, channel catfish,etc.)
• A second family in the phylum Chordata (fish, amphibian, etc.)• A crustacean (e.g., cladoceran, copepod, ostracod, isopod, amphipod,crayfish, etc.)
• An insect (e.g., mayfly, dragonfly, damselfly, stonefly, caddis fly,mosquito, and midge)
• A family in a phylum other than Arthropoda or Chordata (e.g.Rotifera, Annelida, Mollusca, etc.)
• A family in any order of insect or any phylum not alreadyrepresented
• Algae• Higher Plants
Taking into account the results of the literature review andconsidering criteria such as availability of a standard test guidanceand the acceptance of test data for specific species in other metal risksassessment reports, the following organisms were selected as testspecies for chronic toxicity testing: the cladocerans Daphnia magnaand Ceriodaphnia dubia, the fish Pimephales promelas and Oncor-hynchus mykiss, the amphibian Xenopus laevis, the insect Chironomusriparius (midge), the rotifer Brachionus calyciflorus, the snail Lymnaeastagnalis, the green alga Pseudokirchneriella subcapitata, and thehigher plant Lemna minor (duckweed).1
Chronic toxicity tests were conducted at three testing facilities:Laboratory of Environmental Toxicology at Ghent University (UGent,Belgium), the Chilean Mining andMetallurgy Research Center (CIMM,Santiago, Chile), and Parametrix Environmental Research Lab (Albany,Oregon). Testing facilities were selected based on their previousexperience with chronic metal toxicity and with the chronic toxicitytesting procedures that were assigned to these laboratories. For somespecies the tests were conducted in duplicate at different testingfacilities. UGhent performed the tests with X. laevis, C. riparius, B.calyciflorus, L. stagnalis, P. subcapitata, D. magna, and C. dubia. CIMMconducted tests with P. subcapitata and D. magna. Parametrixconducted tests with the fathead minnow (P. promelas) and withthe rainbow trout (O. mykiss).
The chronic tests with D. magna and the green alga P. subcapitatawere conducted at two testing facilities (UGent and CIMM).Preliminary experiments with the algae, however, indicated asignificant difference between the outcomes of both laboratories. Itwas therefore decided to exchange both the test medium (OECD-medium; OECD, 2006a) and algal strain between the two laboratories.Four algal tests were conducted in each testing facility with each testrepresenting a different algal strain and test medium combination.This test design allowed the designation of observed differencesamong effect levels to the algal strain, the test media, or other lab-specific conditions, and resulted in comparable and consistent resultsbetween the two laboratories.
2.4. Test procedures — general information
With the exception of L. stagnalis tests, chronic toxicity experi-ments adhered to internationally accepted standard testing protocols(i.e., OECD, EPA, ASTM, or APHA). No internationally accepted testingprotocols are currently available for L. stagnalis; therefore, method-ology reported by De Schamphelaere et al. (2008) was followed. Adetailed overview about origin of the test organisms, test media, testdesign, exposure duration, test conditions and endpoints recorded areprovided in Annex 1. The test substance was provided by theInternational Molybdenum Association (sodium molybdate dihy-drate; CAS number: 10102-40-6, EINECS 231-551-7; Lot/batch No.:43006L with expiration date 31-12-2009). The salt was a solid, whitecrystalline powder reported to have an analytical purity of 99.9%
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(dihydrate form) based on an analysed Mo-content of 39.6%. The saltwas stored in dry and dark conditions (20 °C±5 °C).
Some general principles essential for generating high quality datawere applied to all tests. Dissolved molybdate levels were measuredand reported effect levels (NOECs, EC10-levels) were based on thesemeasured values. Samples for dissolvedmolybdate (test initiation andtermination, filtered through 0.45 μm)were taken from all treatmentsand measured by ICP-MS or flame Atomic Absorption Spectropho-tometry (USEPA Method 243.1). Physicochemical parameters that arerelevant for a correct assessment of chronic metal toxicity weremonitored before and during the exposure period (e.g., temperature,pH, hardness, and dissolved oxygen content), and steps were taken toensure that variation of these parameters during the exposure periodremained within acceptable boundaries. Finally, criteria for testacceptability were met in each specific test (e.g., growth rate criteria,reproduction criteria, survival criteria, etc.).
2.5. Data treatment/data analysis
In some cases raw biological data (i.e., recorded observations in thedifferent replicates) needed to be treated before calculation of EC10
values. This was the case for L. minor, L. stagnalis, B. calyciflorus, andboth fish tests. For L. minor the growth rate was calculated from theinitial frond number and the frond numbers recorded on days 2, 4, and7 of the test. Following OECD Test Guideline 221 (OECD, 2006b), thegrowth rate in each replicate of each treatment was calculated as theslope of the linear regression of the natural logarithm of the frondnumber versus time. The same principle was applied to calculate thelength and biomass based growth rate of L. stagnalis and thepopulation growth rate of B. calyciflorus. Fish survival data weretransformed by arcsine square root prior to analysis. Beforeanalyzing the fish data, normality and homogeneity assumptionsof survival and weight were evaluated by the Shapiro–Wilk's testand Bartlett's test, respectively (p≤0.01). Because the data satisfiedthe assumptions of normal distribution and homogeneity ofvariance, ANOVA followed by Dunnett's multiple comparison testwas used to compare (p=0.05) organism performance in theexperimental treatments with that observed in the control. Forderivation of the No Observed Effect Concentration (NOEC) orLowest Observed Effect Concentration (LOEC) in the other tests theJonkheere–Terpstra trend test (Lehmann, 1975) was always used. Inshort, this statistical test calculates p-values for the whole dataset(all concentrations); when a statistical significant trend is noted(pb0.05) concentrations are eliminated from the dataset (from highto low) until the trend becomes statistically insignificant (pN0.05).At this point the highest remaining concentration is considered theNOEC. All hypothesis testing was conducted one-sided at the α levelof 0.05 as recommended by OECD (2005). Hypothesis testing wasperformed with SPSS16® software (SPSS Inc., Chicago, Illinois, USA).However, if standard deviations were significantly positivelycorrelated with the means, the non-parametric Mann Whitney Utest with Bonferroni–Holm correction was applied for inferringstatistical differences among control andmolybdate treatments. Thismethod follows the recommendations given in OECD (2005).
NOECs are reported as dissolved concentrations measured at thestart of the test.
Concentrations were measured at the start and—where possible —
at the end of the test. As concentrations had never decreasedmore than10% compared to the initial concentration, the EC10s and 95% confidenceintervals (CI) were calculated based on the dissolved molybdate-concentrations that were measured at the start of the test, using astandard log-logistic concentration–response model unless notedotherwise. The reported effect concentrations therefore refer to theelementalmolybdenum-content in the testmedia (EC10s are expressedasmgMo/L). Calculationswere performed using Statistica® software orToxicity Relationship Analysis Program (TRAP) from the U.S. EPA
National Health and Environmental Effects Research Laboratory(NHEERL).
3. Results
Table 1 gives an overview of the NOEC, LOEC, and EC10 values thatwere generated for the different test species. Algal data are given inTable 2. Table 3 gives an overview of the main test properties for thedifferent species.
In the test with L. minor, the growth rate in the control was0.332 day−1, resulting in a doubling time (calculated following OECDTG221 (OECD, 2006b) of the frond number of 2.1 days. According toOECD 221 (OECD, 2006b), which requires a doubling time b2.5 days,the test is therefore considered valid.
For B. calyciflorus, the mean growth rate in the control was0.734 day−1, which was above the minimum acceptable growth rateof 0.7 day−1 as required by APHA (1998). The test is thereforeconsidered valid.
Control group survival in the two tests with D. magnawas 100% andthe mean control reproduction was 68.3 and 63.0 for the UGhent testand the CIMM test, respectively. Both are higher than the minimumacceptance criteria for survival (80%) and reproduction (60) required byOECDTG211 (OECD, 1998) for a valid test. In theD.magna test atUGhenta non-monotonic dose–response curve was observed (reproductionversus concentration). An initial increase in reproduction was observedin lower exposure concentrations (i.e., up to 85.9 juveniles at 33.8 (mgMo)/L), while at higher concentrations, the reproductive output againdecreased. Since the response was non-monotonic and the variancesamong treatments were not homogenous (Levene's test, pb0.05), theMannWhitney U test with Bonferroni–Holm correction was applied forthe determination of the NOEC and LOEC, following recommendationsbyOECD(2005).Given thesignificant stimulationof reproduction at lowmolybdate concentrations, the non-linear hormesis model of Van Ewijkand Hoekstra (1993) was fitted to the concentration response data forthe determination of the 21 day-EC10.
C. dubia control survival was 100% and the mean reproduction was18.4 juveniles/adult. Both are higher than the minimum survival of80% and reproduction of 15 juveniles/adult required by USEPA (2002)for a valid test.
Nomortality was observed in the control replicates of the test withthe snail L. stagnalis. The biomass growth rate in the control was 7.1%per day, which is consistent with earlier reported growth rates of 4-week old L. stagnalis (De Schamphelaere et al., 2008). These datasuggest that test organisms were “normal” during toxicity testing.
Only 1 of 101 X. laevis embryos in the control died during the test;all other embryos (99%) reached Gosner stage 46 of the embryodevelopment (Gosner, 1960), which is more than the 90% required fora valid test according to the ASTM E1439-91 standard (ASTM, 1998).
For the test with the midge C. riparius there are no standard testvalidity criteria for the endpoints recorded (i.e., survival and growth),but survival in the control was 88% which is consistent with thesurvival acceptability criteria for chronic ecotoxicity test protocols forinvertebrates (D. magna and C. dubia) for which N80% survival isrequired (OECD TG211, OECD 1998; USEPA 2002). Neither survivalnor growth (dry wt) exhibited a monotonic concentration–responserelationship; hence, the Jonkheere–Terpstra step-down test could notbe applied for NOEC/LOEC determination. Furthermore, not all of theconditions necessary for use of a parametric ANOVA approach weremet. For the survival data, variances were not homogenous amongtreatments (p=0.035). For the growth data, the standard deviationswere significantly positively correlated with the means (r=0.78,p=0.022). The EC10 for growth (dry weight), i.e., 121.4 (mg Mo)/L,was determined by fitting the hormesis model of Van Ewijk andHoekstra (1993) to the concentration–response data.
In all fish tests, the control survival in each chamber after thinningof the embryos was higher than 70%, and therefore the tests meet
Table 1Overview of the generated chronic effect levels (EC10, NOEC and LOEC), expressed as mg Modissolved/L for nine test species.
Species Test concentrations (mg Mo/L) Endpoint EC10 NOEC LOEC
mg Modissolved/L
L. minor Total: 26.6; 54.6; 106; 197; 393; 807; 1574 Growth rate (7 days) 241.5(95%CL: 183.6–317.7)
24.7 51.7Dissstart: 22.9; 47.6; 95.8; 191; 393; 794; 1564Dissend: 25.6; 51.7; 103; 204; 410; 845; 1624
B. calyciflorus Diss: 55; 116; 244; 508; 1109; 2301 Population growth rate (48 h) 193.6(95%CL: 49.6–756.3)
244 508
D. magnaU-Gent
Total: 1.2; 19.5; 35.7; 61.6; 109; 197; 329; 576; 1068 Reproduction (21 days) 105.6(95%CL: 914.5–121.8)
112 196Dissstart: 10.8; 19.1; 33.8;60; 112; 196; 325; 569; 1063Dissend: 10.7; 19.4; 35.5; 62.1; 111; 200; 336; 563; n.d.
D. magnaCIMM
Diss: 24.1; 49.9; 89.1; 134.5; 184.5 Reproduction (21 days) 62.8n.d.
49.9 89.1
C. dubia Total: 5.9; 10.6; 17.9; 33.8; 57,8; 100; 183; 325; 557 Survival (7 days) n.d. 177 302Dissstart: 5.3; 10.1; 17.3; 32.4; 55.9; 97.3; 177; 302; 550 Reproduction (7 days) 78.2
(95%CL: 49.8–122.7)97.3 177
Dissend:5.0; 9.8; 17.0; 31.4; 54.6; 95.6; 172; 299; 535L. stagnalis Total: 52.9; 106; 208; 404; 833 Growth rate (length)
(28 days)211.3(95%CL: 173.3–282.5)
200 388Dissstart: 49.0; 101; 200; 388; 808Dissend: 53.4; 102; n.d.; 382; n.d. Growth rate (biomass) (28 days) 221.8
(95%CL: 180.4–272.8)200 388
X. laevis Total: 24.2; 48.7; 97.1; 194; 389 Survival (4 days) 415.4(95%CL: 313.2–550.8)
177 369Dissstart: 22.4; 44.6; 87.3; 177; 369Dissend: 25.1; 50.8; 96.3; 192; 344 Malformation (4 days) 115.9
(95%CL: 34.4–390.5)22.4 44.6
C. riparius Total: 24.8; 50.1; 99.2; 199; 410; 821; 1642 Growth (biomass) (14 days) 121.4(95%CL: 60.9–241.8)
393 794Dissstart: 22.9; 47.6; 95.8; 191; 393; 794; 1564Dissend: 25.6; 51.7; 103; 204; 410; 845; 1624
P. promelas Total: b1; 16.3; 27.4; 54.0; 105.7; 191.9 Dry weight/biomass(34 days)
39.3(95%CL: 32.7–52.6)
27.7 53.0Dissavg: b1; 14.0; 27.7; 53.0;107.6; 203
O. mykiss Total: b1; 120.3; 317.3; 626.6; 1223.3; 2394.9 Biomass 36.9(95%CL: 19.8–68.9)
b121.0 121.0Dissavg: b1; 121.0; 294.9; 579.8; 1146.5; 2328.4
O. mykiss Total: b1; 2.6; 5.3; 13.1; 38.9; 132.4 Biomass 43.2(95%CL: 19.7–94.7)
48.9 152.7Dissavg: b1; 3.6; 6.9; 16.1; 48.9; 152.7
n.d. = not determined.
Table 3Composition of the test media that were used in this study.
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acceptability criteria. In the test with P. promelas the median day tohatch was not different among treatments. No terata (deformities)were noted in exposed organisms during this study and no significanttrend in the survival data was noted. There was, however, a significanteffect in the mean weight per original organism (biomass) so the dryweight data were evaluated using a combined endpoint for lethalityand growth. In both tests with O. mykiss, no effects on the time-to-hatch or egg survival were observed. Significant effects on wet weightper surviving fish, dry weight per surviving fish, length, and conditionfactor, however, occurred in all treatments in the first test.
No effects on dry weight or condition factor were seen in the secondtest. Significant effects on biomass (dry weight per original fish) wereobserved in both thefirst and second testswith LOECvalues of 121.0 and152.7 (mg Mo)/L (dissolved fraction), respectively.
The 72 h-ErC10 values generated in the eight growth inhibition testsare summarized in Table 2. A difference of a factor of 5.98 between thelowest and the highest chronic effect levelwas observed. Exchange teststhat were performed with the algal strain received from CIMM resultedin four 72 h-ErC10 values (ErC10: EC10 based on growth rate) rangingbetween 61.2 and 88.7 mg Mo/L, i.e. a 1.4-fold difference between the
Table 2No effect concentrations (ErC10-levels) of the interlaboratory exchange programbetween CIMM and UGent.
Test medium 72 h-ErC10 (mg Mo/L) (+ −95%CL)
P. subcapitata CIMM P. subcapitata UGent
OECDCIMM, tested at CIMM 88.7 (69.2–106.0) 62.5 (2.5–133.8)OECDUGent, tested at CIMM 77.3 (58.1–95.5) 99.3 (63.5–137.5)OECDCIMM, tested at UGent 61.2 (36.5–102.7) 318.6 (278.9–363.9)OECDUGent, tested at UGent 72.6 (48.3–109.5) 366.2 (294.4–455.5)Mean 75.0 (CV: 11,4%) 211.7 (CV: 153.0%)Geomean 74.3 164.0
lowest and highest value. This difference can be considered within therange of natural biological variation among ecotoxicity tests conductedwith the same species and same test substance. These four effect levelsalso suggest that the origin of the test medium or the test facilitycharacteristics had little or no influence on the outcome of the test.Comparable results (62.5–99.3 mg Mo/L) were also obtained with theUGent strain tested at CIMM. The tests conducted with this strain atUGent, however, produced 72 h-ErC10-levels that were approximately afactor of 4 to 5 higher than the other six tests, but were in line with thetest result generated in the range-finding test at the same testing facility,using the same strain (72 h-ErC10 of 283.8 mg Mo/L, data not furtherdiscussed).
Table 4 gives an overview of the (open) literature data and theirrespective Klimisch scoring. No useful data were identified forprimary producers (algae). For the primary and secondary consumers,
Species Testduration
Endpoint pH mgCa/L
mgMg/L
Pimephales promelas 34 days Biomass 7.5±0.05 22.3 10.4Oncorhynchus mykiss 78/84 days Biomass 7.4±0.1 18.3 8.6Pseudokirchneriellasubcapitata-CIMMstrain
72 h Growth rate 8.0–8.1 4.9 2.9
Ceriodaphnia dubia 7 days Reproduction 7.6–7.9 Hardness:180mg/L CaCO3
Daphnia magna 21 days Reproduction 7.4–8.2 27.9–80.1 12.2–24.2
Xenopus laevis 4 days Malformation 7.8 19.4 15.1Chironomus riparius 14 days Growth 6.9–7.1 14 12.1Brachionus calyciflorus 48 h Reproduction 7.5 14 12.1Lymnaea stagnalis 28 days Length 7.8–8.2 40.1 9.7
Table 4Overview of aquatic chronic toxicity data for molybdate in open literature and in industry reports.
Species Mo-salt Effect level (mg Mo/L) Quality label Reference
Algae (primary producers)Scenedesmus subspicatus NH4-molybdate 72 h-NOEC: 25 K3 HRC (1994a)Scenedesmus subspicatus Na-molybdate 72 h-NOEC: 12.5 K3 HRC (1994b)Scenedesmus subspicatus Mo-trioxide (tech) 72 h-NOEC: ≥100 K3 HRC (1994c)Scenedesmus subspicatus Mo-trioxide (tech) 72 h-NOEC: ≥100 K3 HRC (1994d)Pseudokirchneriella subcapitata Na-molybdate 72 h-NOEC: 4.6 K3 HRC (1996)
Invertebrates (primary consumers)Daphnia magna Na-molybdate 21 day-NOECmort: 373a K1 GEI (2009)
21 day-NOECrepr: 136a K1Daphnia magna Na-molybdate 21 day-NOEC: 50 K3 Diamantino et al. (2000)Daphnia magna Mo-trioxide 21 day-EC10,rm: 6.98 K3 Kimball (1978) (unpublished)
21 day-NOECrm: 4.41 K3Ceriodaphnia dubia Na-molybdate 21 day-NOECmort: 159a K1 GEI (2009)
21 day-NOECrepr: 38.5b K2Ceriodaphnia dubia Na-molybdate 8 day-NOEC: 17c K2/K3 Naddy et al. (1995)
Fish (secondary consumers)Oncorhynchus mykiss Not reported 28 day-LC50: 0.73 K3 Birge (1978); Birge et al. (1980)Oncorhynchus mykiss Na-molybdate 32 day-NOEC: 200 K1 Davies et al. (2005)
32 day-NOEC: 750 K1Oncorhynchus mykiss Na-molybdate 1y-NOEC: N17 K2 McConnell (1977) and anonymous
(18 m-NOEC: N18.5) K2Oncorhynchus mykiss Na-molybdate 60 day-NOECmort: N30 K3 McDevitt et al. (1999)Pimephales promelas Na-molybdate 28 day-EC10,mort: 616.1a K1 GEI (2009)
28 day-EC10,gr: 90.9a K1Carassius auratus Not reported 28 day-LC50: 60 K3 Birge (1978); Birge et al. (1980)Oncorhynchus clarki Na-molybdate 30 day-NOEC: N87.8 K3 Pickard et al. (1999)Oncorhynchus kisutch Na-molybdate 20w-NOEC: N19.5 K2 Ennevor (1993)
Amphibians (secondary consumers)Gastrophryne carolinensis Not reported 28 day-LC50: 0.79 K3 Birge, 1978; Birge et al., 1980
K1: Klimisch 1, reliable.K2: Klimisch 2, reliable with restrictions.K3: Klimisch 3, not reliable.
a Geometric mean of 2 values.b 7 day-EC20/2.c 8 day-IC12.5/2.
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however, Klimisch 1-data were available. It should be noted that K3-data are considered not useful for PNEC-derivation within aregulatory context, but the information in these publications maystill have scientific merit.
4. Discussion
4.1. Evaluation of the generated data
Chronic effect levels for the ten tested species (Tables 1 and 2) werebetween 39.3 mg Mo/L (lowest EC10 for P. promelas; test conducted byParametrix) and 241.5 mgMo/L (EC10 for L.minor),which is a differenceof a factor of 6.15 between the most and the least sensitive species. Theranking in sensitivity of tested species, frommost to least sensitive, is asfollows: P. promelasNO. mykissND. magna (CIMM)NP. subcapitata(CIMM)NC. dubiaND. magna (UGent)NX. laevisNC. ripariusNP. subcapi-tata (UGent)NB. calyciflorusNL. stagnalisNL. minor.
It is noteworthy that the organisms like fish, cladocerans and algae,which are internationally accepted standard test organisms for settingwater quality criteria, andwhich arepreferred for environmental hazardclassification purposes (e.g., the European CLP-regulation EC1272/2008), are among the most sensitive organisms in this ranking. As faras the algal tests are concerned, a marked difference is noted betweenthe tests that were conducted at UGhent with their own, specific strainand the six remaining results (including two tests with the same strainat CIMM). A conclusive explanation for this difference is currently not athand, but the reliability of the two higher values are confirmed by the72 h-ErC10 of 283.8 mg Mo/L derived from the preliminary test (valid
OECD test, effect levels based on measured Mo concentrations). Thesedata suggest that the CIMM-algal strain may be more sensitive than theGhentUniversity strain, and it was therefore decided that only the 72 h-ErC10-values obtained with the former strain should be taken intoaccount for the derivation of a NOEC for P. subcapitata. The geometricmean of these four values is 74.3 mg/L, and this conservative value isconsidered sufficiently protective for the different tested strains for thisalgal species.
From Table 4 it can be concluded that prior to the studies reportedherein, few reliable chronic no-effects data for molybdate wereavailable. GEI (2009) investigated long-term effects of molybdate to D.magna, C. dubia, and P. promelas. Chronic toxicity tests, performed induplicate,wereperformed according to international guidelines (ASTM-Guidelines), using sodiummolybdate as the test substance. The 21 day-EC10,survival value (geometric mean of 2 data points) for D. magna was200 mg Mo/L (145.3–275.4 mg Mo/L); based on reproduction the21 day-EC10 (geometric mean of two data points) was 108.0 mg Mo/L(106–110 mgMo/L). This value is almost identical to the 21 day-EC10 of105.6 (mg Mo)/L that was generated by UGent for this test organism.
For C. dubia, the 7 day-EC10,survival-value (geometric mean of twodata points) was 187.9 mg Mo/L (161.5–218.6 mg Mo/L); based onreproduction the 7 day-EC10 (geometric mean of two data points) was50.8 mg Mo/L (45.6–55.5 mg Mo/L). Again, this value is consistentwith the EC10 generated at UGent for this test organism and endpoint;the difference between the GEI (2009) value and the UGent value is afactor of 1.54 (78.2 mg Mo/L vs. 50.8 mg Mo/L).
Other authors also have reported on chronic toxicity of molybdateto cladocerans (Diamantino et al., 2000; Kimball, 1978; Naddy et al.,
Table 5Overview of most sensitive species-specific EC10-values for molybdate in thefreshwater environment.
Species End parameter NOEC/EC10-value(mg Mo/L)
Referencestudies
Oncorhynchus mykiss Biomass 43.2 This studyPimephales promelas Biomass 39.3; 90.9
geomean: 60.2This study;GEI (2009)
Ceriodaphnia dubia Reproduction 50.8 : 78.2geomean: 63.0
This study;GEI (2009)
Pseudokirchneriellasubcapitata-CIMM strain
Growth rate 61.2; 72.6; 77.3; 88.7geomean: 74.3
This study
Daphnia magna Reproduction 62.8; 105.6; 108.0geomean: 89.5
This study;GEI (2009)
Xenopus laevis Malformation 115.9 This studyChironomus riparius Growth/biomass 121.4 This studyBrachionus calyciflorus Population
growth rate193.6 This study
Lymnaea stagnalis Growth rate/length
221.3 This study
Lemna minor Growth rate 241.5 This study
Fig. 1. Species sensitivity distribution and HC5,50% derivation for molybdate in theaquatic environment.
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1995). Although a thorough evaluation of these data by Heijerick(2009) — applying the criteria set by Klimisch et al. (1997) — hasshown that reported no-effects levels from these studies should notbe considered for regulatory purposes, the “degree of toxicity” ofmolybdate is generally consistent with the findings of the currentstudy. A concise summary of the main arguments that resulted in aK3-classification are provided in Annex 2.
Diamantino et al. (2000) performed a D. magna chronic toxicitytest with sodium molybdate, and both total growth and reproductionwere identified as the most sensitive endpoints with a nominal NOECof 50 (mg Mo)/L for both endpoints. This NOEC-value confirms theNOEC that was reported by CIMM (49.9 (mgMo)/L) for this organism,and is approximately a factor of two below the NOEC that wasdetermined by UGhent.
The result of the Kimball study (1978, unpublished) for D. magna,on the other hand, is more than one order of magnitude below EC10-levels that are reported by UGhent and CIMM, and below the effectslevels that are reported by Canton et al. (2006). An in-depth evaluationof the raw data by Heijerick (2009) led to the conclusion that theoverall health condition of the D. magna batch that was used in theseexperiments could be seriously questioned (see Annex 2). It is alsonoteworthy that this study was rejected by Netherlands NationalInstitute for Public Health and the Environment (RIVM) as a datameeting the acceptability criteria for setting a water quality standardfor molybdate (Van Vlaardingen et al., 2005; Van Vlaardingen andVerbruggen, 2009).
Naddy et al. (1995) examined the sub-lethal interactive effects ofmolybdate (as sodium salt of molybdate) on C. dubia using the three-brood static renewal toxicity test. The end parameters calculated inthis study were 8 day-IC50, 8 day-IC25 and 8 day-IC12.5, and the effectlevels associated with these end parameters were 79.7 (mg Mo)/L,47.5 (mg Mo)/L and 34.0 (mg Mo)/L, respectively. According to theguidelines provided in the former Technical Guidance Document forNew and Existing Substances (TGD) (EC, 2003) and in Chapter 10 ofRIP 3.2 (ECHA, 2008) the default value of ECX divided by 2 (with Xsituated between 10 and 20% effect) can be used as an alternative forthe NOEC in the absence of a bounded NOEC. In this specific situationthis methodology would lead to an estimated 8 day-NOEC of 17 (mgMo)/L (IC12.5 divided by 2). Comparison between the Naddy et al.(1995) data and the data that were generated by UGent shows anacceptable agreement between both studies; i.e., the differencebetween the 8 day-IC12,5 and the 7 day-EC10 is a factor of 2.3.
Several literature data points were identified for the rainbow trout.A very low 28 day-LC50 of 0.73 (mg Mo)/L was reported by Birge(1978) and Birge et al. (1980), and efforts to reproduce this low value(Davies et al., 2005) resulted in markedly higher effect levels formortality after 28 days (28 day-NOEC values of 200–750 (mg Mo)/L).In addition,McDevitt et al. (1999) also reported a 60 day-NOECmortality
of N60 mg/L for O. mykiss, representing a second finding that does notsupport the very low Birge-value. Results of a third experiment werealso similar to the Davies et al. (2005) and McDevitt et al. (1999)findings: McConnell (1977) reported an unbounded NOEC of N17 (mgMo)/L for an exposure period that was markedly longer (one year).UnboundedNOECs that are situatedwithin the sameorder ofmagnitudewere found for two other trout species, namely cutthroat trout O. clarki(30 day-NOEC of N87.8 (mgMo)/L) and coho salmonO. kisutch (30 day-NOEC ofN19.5 (mg Mo)/L) (Pickard et al., 1999; Ennevor, 1993). Allthese findings indicate that the value that was put forward by Birge(1978) and Birge et al. (1980) is an outlier, and should therefore not beconsidered for regulatory purposes (e.g., water quality criteria setting).
The newly generated data by Parametrix (EC10,growth of 43.2 mgMo/L) are also consistent with most of these findings; although thisvalue is more or less a factor of 5–19 lower than the Davies NOECs, itshould be taken into account that Davies et al. (2005) only evaluatedmortality, whereas the Parametrix investigation found growth to bethe more sensitive parameter.
Literature data for other fish species are limited to the goldfish(Carassius auratus) and the fathead minnow (P. promelas). Thegoldfish data are reported in the Birge studies which were previouslycategorized as unreliable. The EC10-level for P. promelas generated byGEI (2009), on the other hand, is considered as a reliable data point.This EC10 value was about a factor of two higher than the EC10 thatwas generated by Parametrix (39.3 (mg Mo)/L vs. 90.9 (mg Mo)/L).
4.2. Derivation of a species sensitivity distribution
A species sensitivity distribution (SSD) has been developed for theassessment of molybdate in the freshwater compartment, using thereliable species-specific chronic toxicity effect levels that have beenidentified in Tables 1, 2 and 4. An overview of these species-specific datais given in Table 5. All toxicity tests were performed using sodiummolybdate as the test substance, and only the most sensitive endparameters were taken into account.
Fig. 1 presents the SSD that was fitted though the ten species-specific no-effect levels that were identified. Using the softwarepackage BestFit, a Log-Normal Distribution was defined as the optimaldistribution. The HC5 and HC5,50% (±95%CL) that were associatedwiththis distribution were 39.5 mg Mo/L and 38.2 mg Mo/L (95%CL: 18.7–57.3 mg Mo/L), respectively. The HC5,50% is the median 5th percentilewith 5%–95%-confidence interval. This confidence interval is calcu-lated using a Monte Carlo analysis on the generated distribution(2000 simulations) and is based on the selection of random samples ofmodel input parameters according to the respective assignedprobability distribution. The outcome of this analysis allows thederivation of the HC5,50% with 5%–95% confidence interval. According
5368 K.A.C. De Schamphelaere et al. / Science of the Total Environment 408 (2010) 5362–5371
to TGD (EC, 2003) and ECHA (2008) Guidance this parameter servesas the reference point for final PNEC-setting.
Compared to other metals that have been subjected to amandatory risk assessment (RA) under European Union CouncilRegulation 793/93/EEC (e.g., Zn) or that conducted a voluntary riskassessment (e.g., Cu), molybdenum as molybdate appears to be a metalthat shows low chronic toxicity towards the aquatic environment. Forinstance, the range of PNEC values for zinc was from 22.1 to 46.1 μgZndissolved/L (range representing bioavailability in different EU-riverbasins) (Van Sprang et al., 2009). These PNECs took all reliable chronicecotoxicity data thatwere present in the Zn-RA report into account. ThePNEC for antimony that was extracted from the last publicly availabledraft risk assessment report under Regulation 739/93/EC, and which isalmost finalized, is 113 μg Sbdissolved/L. The PNEC value for copperdirectly extracted from thefinalized European RiskAssessment report is7.8 μg Cudissolved/L. These PNEC-values are several orders of magnitudebelow the lowest EC10 for molybdenum (as molybdate).
Lemna minor Brachionus c
Testing facility Ghent University Ghent UniveOrigin In-house culture Cysts obtainCulture medium Modified SSI EPA moderaTest protocol OECD TG221 (OECD, 2006b) Cfr APHA 84
Test duration 7 days 48 hTest medium Modified SSI EPA moderaEndpoints Growth rate (based on frond number) Population gTested life stage 2/4 frond colonies 2 h-post hatTested concentrations(nominal)
25 to 1600 mg Mo/L 46, to 2200
Renewal frequency No renewal No renewal# Replicates/conc. 3 8# Individuals/replicate Total of 12 fronds 1Test conditions 100 mL per replicate, 24 °C,
24 L:0 day (85–125 μE m−2 s−1)1 mL per rein darkness
Feeding regime Not applicable At test initia(P. subcapita
Lymnaea stagnalis Xenopus lae
Testing facility Ghent University Ghent UniveOrigin University of Amsterdam UGhent stocCulture medium 2-week acclimation to testing medium Not relevanTest protocol Cfr De Schamphelaere et al., 2008 ASTM E1439Test duration 28 days 4 daysTest medium Modified AFNOR FETAX medEndpoints Survival and growth rate Survival andTested life stage 4-week old juveniles Embryo (gaTested concentrations(nominal)
50 to 800 mg Mo/L 25 to 400 m
Renewal frequency 2× per week Daily renew# Replicates/conc. 8 2 (control: 4# Individuals/replicate 1 25Test conditions 100 mL per replicate, 20 °C, 12 L:12 day
(1000 lx)10 mL per r(10–20 μE m
Feeding regime 60 mg fresh lettuce per snail at everyrenewal (=ad libitum)
No feeding
Daphnia magna
Testing facility Ghent University CIMMOrigin In-house culture Test organisms orig
Aquatic Research OHampton, NH, USA
Culture medium Conditioned city tap water Synthetic hard wate(EPA, 1993; 2002)
Test protocol OECD TG211 (OECD, 1998) OECD TG211 (OECDTest duration 21 days 21 daysTest medium Modified M4-medium Synthetic hard wate
(EPA, 1993; 2002)
5. Conclusion
Chronic toxicity tests were conducted with ten different freshwateraquatic organisms, using sodium molybdate as test substance. Based onthe quality criteria as defined by Klimisch et al. (1997), the chronic no-effect levels taken from these experiments were found to be reliablewithout restrictions (Klimisch 1 data points). Chronic effect levels for thetested species ranged from 39.3 mgMo/L (P. promelas) to 241.5 mgMo/L(L.minor),which is amaximumdifference of a factor of 6.15 among testedorganisms. The data reported herein were generally found to agree withthe limited amount of chronic ecotoxicity data previously reported in theopen literature for this metal. The HC5,50%, taken from the speciessensitivity distribution was 38.2 mgMo/L. Comparison with other metalsrevealed a relatively low degree toxicity of molybdate in the aquaticfreshwater environment. The data that are presented in this paper areintended to become the basis for the derivation of a scientifically-soundPNEC or other water quality standards for the aquatic compartment.
Annex 1. Overview of the chronic toxicity test conditions with sodium molybdate for eight different test species
alyciflorus Ceriodaphnia dubia
rsity Ghent Universityed from Microbiotests NV In-house culturete hard water Conditioned city tap water20 (APHA, 1998) EPA-821-R-02-013 test method 1002.0
(USEPA 2002)7 days
te hard water Conditioned city tap waterrowth rate Survival and reproductionch b24 h old neonatesmg Mo/L 5.6 to 560 mg Mo/L
Daily renewal101
plicate, 24 °C, incubation 15 mL per replicate, 24 °C, 16 L:8 day(10–20 μE m−2 s−1)
tion: 2·106 cells/mLta)
Daily feeding with a mixture of YTC(12 mg solids/L) and P. subcapitata(2·105 cells/mL)
vis Chironomus riparius
rsity Ghent Universityk In-house culturet EPA moderate hard water-98 (ASTM, 1998) OECD TG218 (OECD, 2004)
14 daysium EPA moderate hard watermalformation Survival and growth
strula) 48 h-old larvaeg/L 25 to 1600 mg Mo/L
al 2× per week) 5 (survival) or 10 (growth)
10eplicate, 24 °C, 12 L:12 day−2 s−1)
250 mL per replicate (+quartz sand as substrate),20 °C, 16 L:8 day (1000 lx)
during the test period Tetramin®, daily feeding. 0.25 mg/larva(first week), 0.50 mg/larva (second week)
Pseudokirchneriella subcapitata
Ghent University CIMMinated fromrganisms,
In-house culture, pre-cultureof exponential growing algalcells
In-house culture, obtainedfrom Aquatic ResearchOrganisms, Hampton, NH, USA
r OECD-algal growth medium OECD-algal growth medium
, 1998) OECD TG201 (OECD, 2006a) OECD TG201 (OECD, 2006a)72 h 72 h
r OECD-algal growth medium OECD-algal growth medium
(continued)
Daphnia magna Pseudokirchneriella subcapitata
Endpoints Survival andreproduction
Survival andreproduction
Growth rate (+biomassgrowth)
Growth rate (+biomassgrowth)
Tested life stage b24 h old neonates b24 h old neonates Exponential growing algalcells
Exponential growingalgal cells from a 4-dayold preculture
Tested concentrations(nominal unlessmentioned differently)
10 to 1000 mgMo/L
2.4 to 184.5 mg Mo/L(measured and dissolved)
6 to 61,000 mg Mo/L 32 to 320 mg Mo/L(20 to 400 mg Mo/L)a
Renewal frequency Static renewal; 3× per week Static renewal; minimum 3× perweek
No renewal of test medium No renewal of test medium
# Replicates/conc. 10 10 3 3# Individuals/replicate 1 1 1·104 cells/mL 1·104 cells/mLTest conditions 50 mL per replicate, 20 °C,
16 L:8 day (10–20 μE m−2 s−1)50 mL per replicate, 20±1 °C,16 L:8 day (10–20 μE m−2 s−1)
50 mL/replicate, 24 °C, 24 L:0 day (85–125 μE m−2 s−1)
100 mL/replicate, 22±2.0 °C,24 L:0 day(60–120 μE m−2 s−1).Erlenmeyers placedon an orbital shaker(100 cycles/min)
Feeding regime A 3:1 mixture (cell number basis) ofP. subcapitata and Chlamydomonasreinhardtii; daily feeding; 250 μg,500 μg, 750 μg dry wt in weeks 1, 2,and 3, respectively
A 3:1 mixture (cell number basis) ofP. subcapitata and Chlamydomonasreinhardtii; Each organism received16·106 cells/cup during 21 days
Not applicable Not applicable
aTest conducted with algae from Ghent University.
Annex 1(continued)
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Annex 2. Overview of unreliable (Klimisch 3) chronicecotoxicological data for regulatory purposes
A2.1. Birge (1978) and Birge et al. (1980)
These authors investigated long term toxicity ofmolybdenum for thetoad Gastrophryne carolinensis.
• Exposure periods started upon fertilization of the eggs and endedat 4 days post-hatching, indicating that test results should beconsidered as chronic tests.
• Test period covering an entire life stage, and tested life stageconsidered to be (the most) sensitive.
• However, test methodology for the rapid-scan egg bioassays waspoorly described.
• The Mo-salt that was used in testing was not reported• Mo-levels were measured but not reported.• Detailed information on the characteristics of the reconstitutedmedia is given in Birge and Black (1977).
Pimephales promelas
Testing facility ParametrixOrigin In-house culture
Culture medium Synthetic moderately hard water, 82 mg/L as CaCO3
Test protocol ASTM (2001) Method E1241-92, Standard Guide for ConductingLife-Stage Tests With Fishes
Test duration 34 daysTest medium Synthetic moderately hard water, 82 mg/L as CaCO3
Endpoints Survival and growth (dry wt and biomass)Tested life stageTested concentrations(nominal)
12.5 to 200 mg Mo/L
Renewal frequency Flow-through (continuous-flow serial diluter)Test vessel Glass aquaria placed in a temperature-controlled water bath, an# Replicates/conc. 4# Individuals/replicate 25Test conditions 2.2 L per replicate, 25°±1 °C, 16 L:8 dayFeeding regime 0.15 ml/chamber brine shrimp nauplii 2× day increasing by 0.0
as a function of growth
• Physicochemical parameters were monitored directly (pH, tem-perature, hardness, dissolved oxygen, conductivity, flow rate andtoxicant concentration), but not reported.
• The reported end parameters (LC1,50) are not relevant for the riskevaluation of chronic exposure levels, and with the informationprovided in the two papers it was not possible to derive NOEC- orEC10-values (concentration-specific data not given and the concen-tration-effects relationship is not reported).
• Chronic toxicity data that were reported in these papers for othermetals were rejected for risk/hazard assessment purposes incompulsory and voluntary EU-risk assessment reports based onsimilar arguments (e.g., Cu, Zn, Ni, and Pb).
A2.2. Diamantino et al. (2000)
Results of a Daphnia magna chronic toxicity test with sodiummolybdate was presented in this publication.
• Test procedure followed standard guidelines (static renewal,21 days exposure, six treatment concentrations+control).
Oncorhynchus mykiss
Test #1 Test #2
ParametrixTest organisms originated from a commercial supplier, Trout Lodge, Sumner,WA, USASynthetic moderately hard water,86 mg/L as CaCO3
Synthetic moderately hard water,108 mg/L as CaCO3
Early ASTM (2001) Method E1241-92, Standard Guide for Conducting Early Life-Stage Tests With Fishes84 days 78 daysSynthetic moderately hard water,86 mg/L as CaCO3
Synthetic moderately hard water,108 mg/L as CaCO3
Newly fertilized embryos125 to 2000 mg Mo/L 8 to 128 mg Mo/L
d holding approx. 2.2 L in which glass embryo cups were constructed4 450 502.2 L per replicate, 10±1 °C, 16 L:8 day
5 ml 0.15 ml/chamber trout chow slurry 2× day increasing by 0.05 ml as afunction of growth
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• No information on physico-chemistry during the test period or atthe end of the test was reported (e.g., pH),
• The publication only mentions nominal concentrations with noindication that Mo-levels were measured before or during the testperiod.
• Raw data were not provided; therefore no EC10-levels could bedetermined
A2.3. Huntington Research Centre Study Reports (1994, 1996)
Results of algal inhibition tests with Scenedesmus subspicatus andSelenastrum capricornutum were reported for different Mo-salts
• Reported effects levels are based on nominal levels of themolybdenum salt
• Mo-levels were not measured at the specific request of the sponsor• Some tests were limit tests (i.e., only one concentration as tested)• No decreasing trend in growth rate with increasing Mo-levels isobserved.
A2.4. Kimball-study (1978, unpublished)
Results of a D. magna chronic toxicity test with sodium molybdatewas presented in this publication.
• Report has not been peer-reviewed (unpublished manuscript)• This chronic D. magna study was also rejected and not used byNetherlands National Institute for Public Health and the Environ-ment (RIVM) as data source for setting a water quality standard formolybdenum (Van Vlaardingen et al., 2005; Van Vlaardingen andVerbruggen, 2009).
• Endpoint, end parameter and exposure period are not in line withinternational guidelines for chronic testing with D. magna, andtherefore the data cannot be considered for risk assessmentpurposes (28 day-LC50).
As the raw data were provided in Annex of the manuscript, a21 day-EC10, of 4.3 (mgMo)/L was derived. However, the reliability ofthis value was found to be highly questionable as the followingfindings were observed upon a thorough screening of all raw data inKimball (1978, unpublished):
• Excessive control mortality during the last period of the exposureperiod in several tests. This could point to a poor health condition ofthe organisms in general, resulting in effects on reproduction andsurvival that cannot be attributed solely to increased metal levels,even in tests where no excessive control mortality was noted (e.g.,test with molybdate). A poor health condition can be a confoundingfactor resulting in hypersensitive organisms and molybdate effectlevels that do not reflect the true toxicity of molybdate to a healthycommunity of daphnids.
• Reduced reproduction was noted in control organisms during thelast week of the Mo-test: number of juveniles was in week 4 wasonly 61% of the amount of juveniles in week 3. This observationsupports the assumption of a “poor health condition” thatmay affectthe observation at increased Mo-levels.
• Effects on reproduction were driven bymortality of the adults in thehighest test concentrations, and not by reduced brood sizes: thefinal brood size before an adult organism died was always similar tothe brood sizes in the control at that time. This is not in line with thefindings by e.g., Ghent University who found significant reduction inbrood sizes before mortality occurred (i.e. a gradual decline of thereproduction rate with increasing Mo-concentration levels).
Based on these findings, Heijerick (2009) concluded that there wassufficient uncertainty associated with the data to classify the Kimballstudy as unreliable for risk and hazard assessment purposes.
A2.5. McDevitt et al. (1999)
The chronic toxicity of sodium molybdate dihydrate was investi-gated, using eyed eggs of the rainbow trout (Oncorhynchus mykiss) asthe test organism.
• Mo-levels were not measured after spiking• Dilution medium is not defined, and pH of the test medium duringthe test is not reported
• Reported effect levels are unbounded values (NOECsurvivalN30 (mgMo)/L).
• No data or statistics are reported on behaviour or deformities.
A2.6. Naddy et al. (1995)
The sublethal interactive effects of molybdate (as sodium salt ofmolybdate) on Ceriodaphnia dubia were investigated in this studyusing the three-brood static renewal toxicity test.
• The test methods that were applied in their research followedstandard guidelines, with test conditions that were similar toculture conditions.
• The molybdate levels in the exposure media were measured, andcalculated effect concentrations are based on measured values.
• The end parameters that were calculated in this study were8 day-IC50, 8 day-IC25 and 8 day-IC12.5 (IC: inhibition concentra-tion), i.e., no NOECs or EC10s. The effect levels associated withthese end parameters were 79.7 (mg Mo)/L, 47.5 (mg Mo)/L and34.0 (mg Mo)/L, respectively.
• According to the guidelines provided in the former TGD (EC, 2003)and in Chapter 10 of RIP 3.2 (ECHA, 2008) the default value of ECXdivided by 2 (with X situated between 10 and 20% effect) can beused as an alternative for the NOEC when these parameters are notreported. In this specific situation this methodology would lead toan estimated 8 day-NOEC of 17 (mg Mo)/L (IC12.5 divided by 2).
A2.7. Pickard et al. (1999)
The chronic toxicity of molybdate using a receiving water andeffluent from a mine in British Columbia was determined in a 30 day-salmonid embryo/alevin test with the rainbow trout (Oncorhynchusclarki).
• An unbounded NOEC is reported (i.e., 87.8 (mg Mo)/L)• The highest tested nominal level was 90 mg/L; it is thereforeassumed that 87.8 (mg Mo)/L is a measured value
• Dilution media are not defined• Physico-chemistry of the test medium during the test is notreported
• Test methodology is poorly described.• No data or statistics are reported.
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