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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
Study period:
2013-10-28 until 2014-04-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0 (fresh media), 2, 5, 7, 9, 12, 14, 15, 19 (fresh and old media) and 21 (old media). Additional samples were also taken from Day 2 onwards for further analysis should it be required.
- Sample storage conditions before analysis: The initial test samples were stored frozen prior to analysis, whilst the definitive test samples were analyzed on the day of receipt (excluding the Day 19 definitve test samples which were stored frozen prior to analysis). Samples were taken of the control and each replicate loading rate for chemical analysis. Duplicate samples were also taken and stored frozen should further analysis be required.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Initial test: Based on the results of a preliminary range-finding test the following nominal loading rates were assigned to the initial test: 1.0, 3.2, 10, 32 and 100 mg/L.For the first, second and fourth media renewal amounts of test item (20, 64, 200, 640 and 2000 mg) were each separately added to the surface of 20 liters of test water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. For each remaining media renewal amounts of test item (10, 32, 100, 320 and 1000 mg) were each separately added to the surface of 10 liters of test water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 47 hours and the mixtures allowed to stand for 1 hour. Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no microscopic particles of test item. Due to the significantly variability in the analytical results obtained from the initial test, additional validation work was conducted to determine whether homogenizing the test item using heating produced more consistent measured concentrations. Once this was complete the definitive test was conducted using a modified preparation method as a precautionary measure with the aim of reducing variability.

Definitive test:
The test item was heated at approximately 60 °C for 80 minutes and shaken by hand prior to each use.
Nominal amounts of test item (10, 32, 100, 320 and 1000 mg) were each separately added to the surface of 10 liters of test water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. For the fifth preparation period the 100 mg/L loading rate was not required, and for the remaining preparation periods the 32 mg/L loading rate was not prepared as these were not required. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 47 hours and the mixtures allowed to stand for 1 hour. Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAFs removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs where applicable. Microscopic observations of the WAFs were performed after filtering and showed no microscopic particles of test item.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: waterflea, 1st instar Daphnia magna
- Source: in-house laboratory culture
- Age of parental stock: not specified (only adult mentioned)
- Feeding during test: each culture was fed daily
- Food type: mixture of algal suspension (Desmodesmus subspicatus) and Tetramin(R) flake suspension

ACCLIMATION
- Acclimation period: no

QUARANTINE (wild caught)
not applicable

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Gravid adult daphnids were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
no
Hardness:
224-270 mg CaCO3/L
Test temperature:
19-23°C
pH:
7.4 - 8.2
Dissolved oxygen:
8.1 - 10.2 mg O2/L
Nominal and measured concentrations:
1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs were used.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test substance as a whole, the results were based on nominal loading rates only. The results of these additional preparations indicate significant variability in the measured concentrations that can be obtained using the standard Water Accommodated Fraction preparation method used for a mixture. Based on these results the definitive test was conducted using an additional precautionary measured of heating the test item to 60 °C for approximately 80 minutes to ensure the visual homogeneity of the test item. However, it was also understood that this measure may have been insufficient to obtain consistent measured concentrations in any repeat testing due to the complex nature of the test item.
Details on test conditions:
TEST SYSTEM
- Test vessel: 150 mL glass flask, covered with plastic lid to reduce evaporation
- Fill volume: 100 mL test preparation
- Aeration: the diluent water only was aerated prior to use, the test vessels were not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency): 3 days
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Test medium: Elendt M7
- Preparation of dilution water: deionized reverse osmosis water
- Culture medium different from test medium: no
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: not mentioned
- Photoperiod: 16 h (8 h darkness with 20 min dawn and dusk transition periods)
- Light intensity: 720-796 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Parental: daily (dead/alive)
- Filial: daily (dead alive)
- Other: unhatched eggs

RANGE-FINDING STUDY (48h)
- Test concentrations: nominal loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes (no immobilization in any concentration)


Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Remarks on result:
other: data not sufficient for calculation
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Remarks on result:
other: data not sufficient for calculation
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
4.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Remarks on result:
other: data not sufficient for calculation
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
reproduction
Remarks on result:
other: data not sufficient for calculation
Details on results:
- Mortality of parent animals: 100 % mortality by Day 8 at loading rates of 32 and 100 mg/L, single mortality in the 10 mg/L loading rate WAF and 20 % mortality in the control and in the 1 mg/L loading rate WAF, no further mortality was observed in these test groups, therefore for the remainder of the test this was thought not to significantly impact the test
- Body length and weight of parent animals: there were statistically significant differences between the control and the 10 mg/L loading rate WAF in terms of length of the daphnids after 21 days exposure
- Type and number of morphological abnormalities: none
- Type and number of behavioural abnormalities: none
- Other biological observations: no unhatched eggs, no dead young, changes in the appearance (smaller and paler than the control) of the Daphnids in the 10, 32 and 100 mg/L loading rate, whilst the 32 and 100 mg/L loading rate were immobilised by Day 8, the 10 mg/L loading rate test group recovered by Day 12 and was not significantly different from the control group in their general color or appearance.
After 21 days in the 10 mg/L loading rate WAF a statistically significant difference from the control and the remaining test groups of producing fewer numbers of live young per adult was seen, however, not in the 1 and 3.2 mg/L loading rate WAF
Reported statistics and error estimates:
The EL50 (immobilization) value on Day 21 was calculated by the geometric mean method using the ToxCalc computer software package (ToxCalc 1999) using the method as follows:
EL50 value = 21C x C
Where:
C1 = concentration showing 0% immobilization
C2 = concentration showing 100% immobilization
If there is no significant immobilization between 0% and 100% immobilization, then the geometric mean of the highest test concentration showing no lethality and the lowest test concentration showing 100% lethality is calculated.
The EL50 (reproduction) value after 21 days was calculated by the maximum-likelihood probit method (Finney 1971) using the ToxCalc computer software package (ToxCalc 1999).
Probit analysis is used where two or more partial responses to exposure are shown.
For the estimation of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) the numbers of live young produced per adult over the duration of the test for the control and each test group were compared using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and the Williams test for differences between treatment means when several dose levels are compared with a zero dose control (Williams 1971) (see Appendix 5). Results from the control and each test group Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using one way analysis of variance incorporating Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1981) and Dunnett’s multiple comparison procedure for comparing several treatments with a control (Dunnett 1955) (see Appendix 5). All statistical analyses were performed using the SAS computer software package (SAS 1999 - 2001)
The MATL was calculated using the geometric mean method as follows:
MATL value = LOEL x NOEL

During an initial test performed, analysis of the freshly prepared 10, 32 and 100 mg/L loading rate WAFs showed measured concentrations of between less than the limit of quantification (LOQ), determined to be 0.0013 mg/L for the analytical method employed, and 25 mg/L. Analysis of the old or expired media for the 10, 32 and 100 mg/L loading rate WAFs showed measured concentrations of between less than the LOQ and 0.011 mg/L. Due to the significant variability in the measured concentrations observed between time points, the duplicate samples were also analyzed. The duplicate analysis of the freshly prepared media showed measured concentrations of between less than the LOQ and 23 mg/L and the duplicate analysis of the old or expired media showed measured concentrations of between less than the LOQ and 0.81 mg/L. The initial test provided significant variability in the measured concentrations for all loading rates and as such was repeated to determine if the variability observed was due to the inherent characteristics of the test item. Analysis of the freshly prepared 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs in the definitive test showed measured concentrations of between less than the LOQ, determined to be 0.0013 mg/L for the analytical method employed, and 5.7 mg/L. Analysis of the old or expired media for the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs in the definitive test showed measured concentrations of between less than the LOQ and 0.13 mg/L. Harlan Study Number: 41204246 Report Page 9 These results indicate significant variability in the measured concentrations obtained for the loading rates in the definitive test which was consistent with the observations in the initial test. This variability may have been due to an inherent characteristic of the test item which caused difficulty in the analysis which, coupled with the sensitivity of the analytical method, caused significant variability in the measured concentrations obtained. It should also be noted that higher measured concentrations were obtained for the range-finding and initial tests, and yet lower toxicity was observed, indicating that the toxicity of the test item was independent of the measured concentrations obtained. In both the initial and definitive test, measured concentrations were observed for the control group of between less than the LOQ and 0.029 mg/L. During the definitive test all precautions against contamination during the test in addition to analytical contamination and instrumentation carry over were taken. This included pre-cleaning of the rotary evaporators and running blank solvent injections prior to running control samples. Duplicate analysis was also performed for each test concentration to improve the reproducibility. However it was still evident that at dose concentrations below the 10 mg/L loading rate the analytical results obtained were inconsistent and variable and no reliability could be given with any significant confidence. On several occasions concentrations were also detected in the controls above that of the LOQ, although in the majority of the cases these were only just above the background LOQ and can be considered as insignificant in real practical terms since they were less than 10 ppb. The LCMS analytical technique used for this analysis was extremely sensitive and had an LOQ of approximately 1 ppb and therefore any slight background contamination or instrument carry over was easily detected. In addition, as no significant immobilization or impairment of reproduction was observed in the control group, this was considered not to impact the test. Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Validity criteria fulfilled:
yes
Conclusions:
The definitive test exhibited significantly lower toxicity values when compared to the initial test. This may have been due to the fact that the method of preparation for the test item was slightly altered as the test item was heated at approximately 80 °C prior to each use to ensure homogeneity in the definitive test. In addition, variable measured concentrations were obtained in both the initial and definitive test. However, this was considered not to impact the outcome of the study as the toxicity appeared to have been independent of the measured concentrations obtained as the initial test exhibited higher toxicity values but higher measured concentrations, whereas the definitive test showed lower toxicity values but lower measured concentrations.
The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental Daphnia generation (P1) was calculated to be 18 mg/L loading rate WAF.
The 21-Day EL50 (reproduction) based on nominal loading rates was 12 mg/L loading rate WAF.
The "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF respectively. The "Maximum Acceptable Toxicant Loading Rate" (MATL) was calculated to be 5.7 mg/L loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
Executive summary:

The study was performed to assess the chronic toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with OECD Guidelines for Testing of Chemicals (2008) No 211, “Daphnia magna Reproduction Test” referenced as Method C.20 of Commission regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects Test Guidelines OPPTS 850.1300 “Daphnid Chronic Toxicity Test”.

Range-finding Test: No immobilization was observed at the nominal loading rates of 1.0, 10 and 100 mg/L. Based on this information nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the initial and the definitive test. Chemical analysis of the 1.0, 10 and 100 mg/L loading rate test preparations at 0 and 48 hours showed measured concentrations of between less than the limit of quantification calculated to be 0.0013 mg/L for the analytical employed and 26 mg/L.

Initial Test : The No Observed Effect Loading Rate (NOEL) for the initial test was determined to be 10 mg/L loading rate WAF and the Lowest Observed Effect Loading Rate (LOEL) for the initial test was determined to be 32 mg/L loading rate WAF. The initial test provided significant variability in the measured concentrations for all loading rates and as such was repeated to determine if the variability observed was due to the inherent characteristics of the test item.

Definitive Test: Mortality (immobilization) occurred predominantly at the highest test loading rates of 32 and 100 mg/L resulting in 100% mortality by Day 8 in these test groups. A single mortality was also observed in the 10 mg/L loading rate WAF test group and 20% mortality was observed in the control and in the 1.0 mg/L loading rate WAF test group. However, as no further mortality was observed in these test groups for the remainder of the test this was thought not to significantly impact the test.

After 21 days the length of each surviving adult was determined. The results showed that there were statistically significant differences (P≤ 0.05) between the control and the 10 mg/L loading rate WAF test group in terms of length of the daphnids after 21 days exposure to the test item. The 32 and 100 mg/L test group data was not included in this analysis as exposure to the test item eliminated all the daphnids prior to Day 21 of the test. In addition, changes in the appearance of the Daphnids in the 10, 32 and 100 mg/L loading rates were also observed whereby they were observed to be smaller and paler than the control group. Whilst the 32 and 100 mg/L loading rate test groups were observed to be immobilized by Day 8, the 10 mg/L loading rate test group were observed to be recovered by Day 12 and not significantly different from the control group in their general color or appearance.

After 21 days there were no statistically significant differences between the control and the 1.0 and 3.2 mg/L loading rate WAF test group in terms of the number of live young produced per adult. The 10 mg/L loading rate WAF test group showed a statistically significant difference (P≤ 0.05) from the control and the remaining test groups after 21 days in terms of producing fewer numbers of live young per adult. The 32 and 100 mg/L test group data was not included in this analysis as exposure to the test item eliminated all the daphnids prior to Day 21 of the test.

Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test. Young were first produced in the control test group on Day 7 of the test. There were no unhatched eggs or dead young in all control and treatment groups surviving to maturation.

The "Lowest Observed Effect Loading Rate" (LOEL) was 10 mg/L as the adults in this test group were significantly smaller (P < 0.05) and produced fewer young than the control group.

The "No Observed Effect Loading Rate" (NOEL) was 3.2 mg/L as there were no significant mortalities (immobilization) observed in the parental generation (P1), the adults were not significantly smaller (P≥0.05) than the control group and there were no significant differences (P≥0.05) in terms of the number of live young produced per adult when compared to the control after 21 days.

Description of key information

The definitive test exhibited significantly lower toxicity values when compared to the initial test. This may have been due to the fact that the method of preparation for the test item was slightly altered as the test item was heated at approximately 80 °C prior to each use to ensure homogeneity in the definitive test. In addition, variable measured concentrations were obtained in both the initial and definitive test. However, this was considered not to impact the outcome of the study as the toxicity appeared to have been independent of the measured concentrations obtained as the initial test exhibited higher toxicity values but higher measured concentrations, whereas the definitive test showed lower toxicity values but lower measured concentrations.

The 21-Day EL50 (immobilization) value, based on nominal loading rates, for the parental Daphnia generation (P1) was calculated to be 18 mg/L loading rate WAF.

The 21-Day EL50 (reproduction) based on nominal loading rates was 12 mg/L loading rate WAF.

The "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF respectively. The "Maximum Acceptable Toxicant Loading Rate" (MATL) was calculated to be 5.7 mg/L loading rate WAF.

Given that the toxicity cannot be attributed to a single component or a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
3.2 mg/L

Additional information