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Ecotoxicological information

Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2019 - XXXX
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
CAS number: 10254-57-6
Appearance: Amber-green liquid
Storage conditions: Controlled ambient temperature (15 to 25 deg. C), in the dark
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the control, solvent control and the test group from the freshly prepared bulk test preparation on Days 0, 6, 13, 20, 27 and 32 and from the old media on Days 1, 7, 14, 21, 28 and 33 (from a single replicate of each treatment group, changing systematically amongst replicates throughout the test). Samples taken from the test solutions throughout the definitive test were prepared with and without centrifugation (40,000g for 30 minutes) prior to analysis in an attempt to assess the dissolved and hence bioavailable test item concentrations. The samples were stored frozen prior to analysis.
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
Preliminary solubility work indicated that it was possible to obtain a testable solvent stock solution of the test item at a concentration of 10 mg/ml in dimethylformamide (DMF). Exposure under dynamic (continuous flow) test conditions using suitable solvent stock solutions throughout the test, prepared with an auxiliary solvent at a maximum concentration of 100 μL/L (final volume), was proposed initially. The measured test concentrations over a 10-day media preparation trial were very inconsistent, however. Based on the results of a second media preparation trial, it was considered appropriate to employ semi-static exposure conditions with test solutions prepared by spiking the test media with a solvent stock solution and dispersing in test water with the aid of magnetic stirring. It was shown during preliminary work that a higher concentration (0.5 mg/L) yielded hazy dispersions and did not appear to significantly increase the levels of dissolved test item. Consistent with other ecotoxicological studies conducted with this test item and the reported water solubility, a nominal concentration of 0.24 mg/L was therefore employed in the definitive exposure.

On Days 0, 7, 14, 21 and 28, a nominal amount of test item (96 mg) was dissolved in DMF and the volume adjusted to 20 mL to give a 4.8 mg/mL solvent stock solution (solvent stocks were shown to be stable for at least 8 days when stored refrigerated in the dark). On Days 0, 1, 2 and 4 to 32, an aliquot (1100 uL) of this solvent stock solution was dispersed in 22 liters of test water with the aid of vigorous stirring with a magnetic stirrer for 5 minutes to give a nominal test concentration of 0.24 mg/L.

The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 uL/L of DMF, the same solvent concentration in the test solution.

After dosing all control, solvent control and the test concentration were observed to be clear, colorless solutions by visual inspection. With the exception of one occasion where the aged test solutions were observed to be slightly cloudy solutions (Day 18 solvent control and 0.20 mg/L test group). All aged control, solvent control and the test concentration were observed to be clear, colorless solutions by visual inspection.
Test organisms (species):
Pimephales promelas
Details on test organisms:
The test was initiated with newly fertilized eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were bred at Covance CRS Research Limited on 14 January 2019 and maintained in dechlorinated tap water.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
33 d
Hardness:
140 to 150 mg/L as CaCO3 (Day 0);
156 to 170 mg/L as CaCO3 (Day 33)
Test temperature:
24 - 26°C
pH:
7.5 - 8.9
Dissolved oxygen:
4.3 mg/L
Nominal and measured concentrations:
Nominal limit concentration: 0.24 mg/L
Geometric mean measured concentration: 0.20 mg/L*
Geometric mean measured concentration (centrifuged): 0.034 mg/L

* basis for effect concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass vessels
- Size of vessel: 1 liter glass vessels used from Days 0 to 14; 5 liter glass vessels used from Days 15 to 33
- Type: Semi-static test conditions
- Aeration: Test vessels aerated via narrow bore glass tubes from Day 12 onwards.
- Renewal rate of test solution (frequency/flow rate): Daily renewal, with the exception of Day 3, when there was no water change to avoid causing premature hatching of the eggs.
- No. of organisms per vessel: twenty eggs
- No. of vessels per concentration (replicates): Four replicate flasks
- No. of vessels per control (replicates): Four
- No. of vessels per vehicle control (replicates): Four

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Laboratory tap water declorinated by passage through an activated carbon filter and a portion of the incoming water softened and then remixed with the main supply.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days.
- Light intensity: observed to be in the range 427 to 561 Lux
Reference substance (positive control):
yes
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
> 0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Hatching and post-hatching survival, body length and wet weight
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Survival or growth
Details on results:
Analysis of the fresh test preparations on Days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.18 to 0.23 mg/L (0.030 to 0.083 mg/L after centrifugation). Analysis of the aged test preparations on Days 1, 7, 14, 21, 28 and 33 showed measured test concentrations to be between 0.10 and 0.27 mg/L (0.0060 and 0.049 mg/L). The measured concentrations in the non-centrifuged samples confirmed that the test system was correctly dosed throughout the test and that concentrations generally remained stable over each 24 hour renewal period, with the exception of Days 13 to 14.

Based on data from preliminary trials in this study and a concurrent surface water mineralization study (Covance Study No. BK38HK), in which the addition of acetonitrile at various stages of sample processing yielded varied recoveries, it appears adsorption of the test item onto the centrifuge tubes may have contributed to the low recoveries within the centrifuged test samples in this study and, therefore, may not reflect actual exposure concentrations within the test system. There were no physical effects noted on the fish (coating of gills, etc.) that may have been attributable to undissolved test item in the test system. The effect concentrations in this study were therefore determined based on the geometric mean measured test concentration of the non-centrifuged test samples (0.20 mg/L).

The number of dead eggs was low throughout the test. The start and completion of egg hatching was observed on Day 5 of the test. There were no sub-lethal effects observed during the test.

Statistical analysis of the hatching data, post-hatch survival data, fish body length, and fish wet weight was carried out for the pooled controls and the 0.20 mg/L test group. There was no statistically significant difference (P>=0.05), between the pooled controls and the 0.20 mg/L test group for hatching, post-hatch survival or fish weight. The 3.1% reduction in body length in the test group was determined to be statistically significant difference (P<0.05) from the pooled controls; however, this reduction was not considered to be a biologically significant effect.

Further evaluation relative to the historical control data confirmed that fish lengths in the treatment group were within the historical control range with the exception of one fish having a lower length. In contrast, a total of twenty-one fish within the control group and eight fish in the solvent control group had longer lengths than the historical control range. Two fish were smaller in the control group and one was smaller in the solvent control group relative to the historical control range. In conclusion, all but one fish exposed to the test item exhibited body lengths within the historical control range; however, group mean fish lengths in both control groups were longer than the historical control range, making the test group appear disproportionately small. As there were no statistically significant effects noted on body weight, the overall reduced mean body length in the test group was considered to be of no toxicological significance.

Reported statistics and error estimates:
Prior to analysis of the test groups, the control and solvent control group data for hatching and post-hatch survival were compared using the using the Chi squared 2x2 Table Test. No significant differences (P>=0.05) were found between the control and solvent control, therefore the test groups were compared to the pooled control groups. Analysis data in the control and test group were compared using the Chi squared 2x2 Table Test to assess for effects on hatching and survival.

Prior to analysis of the test groups, the control and solvent control group data for body length and weight were compared using the Student-t test for Homogeneous Variances. No significant differences (P>=0.05) were found between the control and solvent control, therefore fish growth parameters in the test groups were compared to that of the pooled control groups. Analysis of the fish length and weight data obtained at termination of the test from the pooled control and test group were compared using the Student-t test for Homogeneous Variances, incorporating Levene’s Test on Variance Homogeneity and Shapiro-Wilk’s Test on Normal Distribution.
Validity criteria fulfilled:
yes
Conclusions:
Based on the geometric mean measured concentration, the E(L)C10 and E(L)C50 values were determined to be greater than 0.20 mg/L for hatching, post-hatch survival, body length and wet weight. The No Observed Effect Concentration (NOEC) based on survival or growth was 0.20 mg/L.
Executive summary:

No mortalities or sub-lethal effects were observed following exposure of fathead minnow (Pimephales promelas) larvae to the test material for 33 days (28 days post-hatch).  Based on the geometric mean measured concentration, the E(L)C10 and E(L)C50 values were determined to be greater than 0.20 mg/L for hatching, post-hatch survival, body length and wet weight. The No Observed Effect Concentration (NOEC) based on survival or growth was 0.20 mg/L.

Description of key information

No mortalities or sub-lethal effects were observed following exposure of fathead minnow (Pimephales promelas) larvae to the test material for 33 days (28 days post-hatch).  Based on the geometric mean measured concentration, the E(L)C10 and E(L)C50 values were determined to be greater than 0.20 mg/L for hatching, post-hatch survival, body length and wet weight. The No Observed Effect Concentration (NOEC) based on survival or growth was 0.20 mg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
2 mg/L

Additional information

Upon review of the methylenbis(dibutyldithiocarbamate) acute ecotoxicity data, the NOEC results to aquatic invertebrates (0.052 mg/l) and fish (0.06 mg/l),Daphnia magnaare considered the most sensitive species in terms of aquatic testing. To avoid unnecessary testing the most sensitive species can be chosen as the most appropriate species for long term testing. According to Column 2 of Annex IX of the European Union (EU) Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) 1907/2006 regulation the choice of appropriate testing depends upon the results of the chemical safety assessment. Based on acute aquatic results for methylenbis(dibutyldithiocarbamate)daphnia magnaare the more sensitive species and thus will yield sufficient conservative results to quantify the potential hazards of the test substance while also providing an appropriate hazard assessment. Chronic testing is available with the most sensitive speciesdaphnia magna, therefore, chronic testing of fish is being waived.