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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study conducted under GLP. Exposure concentrations, measured on the basis of total Sn, were converted to and reported as the named substance [MMT(2-EHMA)] using the conversion factor [743.7/118.71] consisting of the Mol weights of the test substance and Sn, respectively. All measured Sn was attributed to the named substance. This approach includes more water-soluble tin impurities and hydrolysis products. The preparation of the WAF allowed both a solubilized portion of the test substance and the hydrolysis products to be present in the study. Analytical repeatability was difficult due to the low solubility of the named substance. This is not unexpected given the low water solubility of the named substance and its instability in water. In the lowest treatment group (1% of a WAF), the test concentrations were below the limit of detection (LOD = 4.5 µg Sn/L), except at t = 12 and t = 16 days in the newly prepared test solutions. Half the LOD (2.3 µg/L) was used to calculate the Sn concentration.
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
During preparation of the stock solution for the first renewel, the magnetic stirring period was 27.5 hours instead of 24 hours; Statistical analysis was performed on parental survival, using Finney; The project number of the most recent study in which th
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations
- Sampling method: at the beginning and end of three intervals of 48 hours (nominal days 0 and 2, 5,and 7 and 12 and 14) and of an interval of 72 hours (nominal days 16 and 19). Undiluted WAF: additionally at the start of the 2nd, 4th, 5th and 7th and 9th interval (nominal days 2,7,9,14 and 19)
- Sample storage conditions before analysis: samples were stored at room temperature
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution with a loading rate of 100 mg/L was prepared and stirred for 24 h after which it was left to stabilise for another 24 h. The water accommodated fraction (WAF) was separated from the undissolved fraction of test substance using pipette force, filtered over a glass filter (GF/C Whatman) and diluted in M7 medium.
- Differential loading: loading rate of 100 mg/L
- Controls: M7 test medium
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): all media were completely clear throughout the test
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain/clone: Daphnia magna
- Justification for species other than prescribed by test guideline: selected as an internationally accepted species
- Source: culture of the performing laboratory
- Age of parental stock (mean and range, SD): <24 h
- Feeding during test
- Food type: algae cells (Chlorella pyrenoidosa)
- Amount: 0.22 - 0.24 mg C/daphnid/day
- Frequency: daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: each new batch was started by placing about 250 newborn daphnids (<3 days old) into 5 litres of medium in an all glass culture vessel. Renewl of the cultures after 7 days of cultivation half of the medium twice a week
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
none
Hardness:
232 to 304 mg CaCO3/L
Test temperature:
19.1 to 20.7 °C (test media) and 18.3 to 20.1 °C (room temperature)
pH:
6.6 to 8.1
Dissolved oxygen:
>6.0 mg/L
Salinity:
not reported
Nominal and measured concentrations:
nominal 0, 1, 5, 10, 50 and 100% of a Water Accommodated Fraction (WAF) prepared at a loading rate of 100 mg/L
measured concentrations: This equals 3.5, 2.9, 11, 21, 111, and 369 micrograms Sn/L (measured) or 22, 18, 67, 134, 694, and 2315 micrograms/L as MMT(2-EHMA) (calculated).
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: all glass
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static
- Renewal rate of test solution (frequency/flow rate): 3 times/week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20
- No. of vessels per vehicle control (replicates): none

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7 medium; prepared in the laboratory according to Elendt (1990)
- Total organic carbon: 4.250 mg/L
- Particulate matter: not reported
- Metals: not reported
- Pesticides: not reported
- Chlorine: not reported
- Alkalinity: not reported
- Ca/mg ratio: not reported
- Conductivity: not reported
- Salinity: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light/8 h dark
- Light intensity: 690-760 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility and mortality and the presence of eggs in the brood pouch every workday; body length at the end of the test; appearance of firs brood when observed; newborn daphnids every workday; presence of unhatched eggs and incidence of immobility when observed.

VEHICLE CONTROL PERFORMED: yes/no- not applicable; no vehicle

RANGE-FINDING STUDY- no range-finder conducted
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.13 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.13 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: reproductive output, mortality, body length
Key result
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.13 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LC50
Effect conc.:
0.305 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: parental survival
Details on results:
- Mortality of parent animals: 10 % (1 % WAF), 10 % (5 % WAF), 10 % (10 % WAF), 100 % (50 % WAF) and 100 % (100 % WAF)
- No. of offspring produced per day per female:
- Body length and weight of parent animals: Average lenght 4.24, 4.44, 4.16, 4.12, not measured, and not measured for the 0, 1.0, 5.0, 10, 50 and 100 % concentrations, respectively. Body length was not measured for the two highest test concentrations, since all parental daphnids had died during the test. Weights were not measured.
- Type and number of morphological abnormalities: aborted eggs were observed in the control and the lowest three concentrations tested. Unhatched eggs could not be attributed to treatment; One daphnid at 1 % of the WAF was considered small on Day 5.
- Type and number of behavioural abnormalities: one immoblie young was produced in each of the 5 and 10 % concentrations. Immobile young could not be attributed to treatment.
- Number of males and females (parental)
- Time to first brood release or time to hatch: day 8
- Egg development time not reported
- Brood size not reported
- Time to sexual maturity not reported
- Effect concentrations exceeding solubility of substance in test medium: the test substance was not completely soluble in test medium; WAF were prepared.
Results with reference substance (positive control):
No reference substance
Reported statistics and error estimates:
For each concentration the results of reproduction were tested for normality and for homogeneity of variance. Furthermore, these data were statistically tested using an ANOVA test followed by a mean comparison test (Bonferroni t-test, Tukey test and Dunnett's test)
As no partial mortality occurred, the LC50 was calculated as AB^1/2, where A and B are the concentrations at which mortality was 10 and 100 %, respectively.

Body length was statistically tested using an ANOVA test followed by a Bonferroni t-test and a Tukey test.

Statistical analysis on the cumulative reproduction of young showed that the reproduction of the daphnids was not significantly reduced up to 134 µg MMT(EHTG)/L, but significantly reduced at 694 µg MMT(EHTG)/L. Mean body length was not statistically reduced at the lowest three concentrations.

METHOD OF CALCULATION: Measured Sn concentrations were used to estimate test substance concentrations by applying a multiplication factor of 6.27 [the ratio of the molecular weights of Sn (118.71 g/mol) and the test substance (743.7 g/mol)]; time weight average exposure concentrations were calculated from the actual Sn concentrations.

In the lowest treatment group, the test concentrations were below the limit of detection (LOD = 4.5 µg Sn/L), except at t = 12 and t = 16 days in the newly prepared test solutions. Half the LOD (2.3 µg/L) was used to calculate the Sn concentration.

Validity criteria fulfilled:
yes
Conclusions:
Exposure concentrations up to 134 µg MMT(EHTG)/L did not induce significant inhibition of the reproductive capacity of the parental daphnids. Exposure to 694 and 2315 µg MMT(EHTG)/L induced complete mortality of the daphnids.
Executive summary:

Parental mortality was 100 % in Daphnia magna exposed to 50 % (0.694 mg/L) and 100 % (2.315 mg/L) of the Water-Accommodated Fraction of MMT(2-EHMA). Reproduction was not affected at concentrations up to 0.134 mg/L (10 % of the WAF). The 21-day LC50 for parental survival was 0.305 mg/L, the overall LOEC was >0.134 mg/L, and the overall NOEC was 0.134  based on measured concentrations. The test was conducted according to OECD Test Guideline 211.

Description of key information

Chronic daphnia: Overall NOEC after 21-days 134 µg/L (OECD 211); 21-day NOEC for reproduction ≥ 134 µg/L; 21-day LC50 for parental survival 305 µg/L; 21-day EC50 for reproduction >134 µg/L

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.134 mg/L

Additional information

The long-term toxicity to aquatic invertebrates was tested in an OECD 211 21-day Daphnia magna reproduction study. The study investigated the effects on survival and reproductive performance. Exposure to concentrations up to 134 µg/L did not induce significant inhibition of the reproductive capacity of the parental daphnids. Exposure to 694 and 2315 µg/L induced complete mortality of the daphnids.