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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:
2021/02/01 - 2021/07/09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
not applicable
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Based on the recommendations of OECD Guidance Document No. 23 (OECD, 2019).
Deviations:
yes
Remarks:
The variation of pH for the control group was 1.6, rather than ≤ 1.5. The variation of temperature was 3.7 ℃ in the range-finding test and 3.6 ℃ in the definitive test, rather than ≤ 2 ℃. These minor deviations will not affect the test integrity.
GLP compliance:
yes (incl. QA statement)
Remarks:
The UK Good Laboratory Practice Regulations (Statutory Instrument 1999 No. 3106, as amended by Statutory Instrument 2004 No. 994)
Analytical monitoring:
yes
Remarks:
Water samples were taken from the control, solvent control and each test group for quantitative analysis. Samples of the fresh test preparations were taken from the bulk test preparations on Days 0, 6, 13 and 20
Details on sampling:
- Concentrations: The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0, 6, 13 and 20 (fresh media) and on Days 1, 7, 14 and 21 (old media). Chemical analysis of the fresh test preparations on Days 0, 6, 13 and 20 showed measured test concentrations to range from 0.0052 to 0.22 mg/L. Chemical analysis of the corresponding aged test preparations on Days 1, 7, 14 and 21 showed measured test concentrations to range from 0.0052 to 0.18 mg/L. Due to the variability of measured test item concentrations between each preparation period, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were calculated to be 0.0066, 0.018, 0.046, 0.099 and 0.14 mg/L.
- Sampling method: From fresh and aged samples. Days 0, 6, 13 and 20 (fresh media) and on Days 1, 7, 14 and 21 (old media)
- Sample storage conditions before analysis: Analysed on the day of sampling or stored frozen until analysis


Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method for the definitive test: Solvent spike preparation
A nominal amount of test item (100 mg) was dispersed in 10 mL of dimethylformamide to give a 10000 mg/L solvent stock solution. An aliquot (200μL) of this solvent stock solution was dispersed in 2 L of test media with the aid of magnetic stirring for approximately 30 minutes to give a concentration of 1.0 mg/L. A sample of the untreated 1.0 mg/L solution was taken for analysis and the any undissolved test item was removed from the remaining preparation by centrifugation at 10000 g for 30 minutes. A series of dilutions was made to give 0.010 and 0.10 mg/L. Samples of to 0.010 and 0.10 mg/L preparations were taken for analysis.

- Controls: The solvent control group was exposed to 100 μL/L of dimethylformamide.
- Chemical name of vehicle: dimethylformamide
- Test concentration separation factor: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.
- Evidence of undissolved material : Following stirring any undissolved test item was removed by centrifugation at 10000 g for 30 minutes to give the required nominal test concentrations of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.
- Other relevant information: For each concentration a single daphnid was placed in 100 mL of the test preparation in 150 mL glass vessels which were then covered with a plastic lid to reduce evaporation. For each test and control group 10 replicate test vessels were prepared. The test vessels were maintained in a temperature controlled room at 18 to 22 C with a photoperiod of 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods for 21 days. The test vessels were not aerated. The diluent water only was aerated prior to use. The test preparations were renewed daily throughout the test. The adult Daphnia were transferred to fresh media by wide-bore pipette before the contents of each vessel were passed through a fine mesh. Young daphnids (live and dead) and any unhatched eggs were collected on the mesh and counted.
Each daphnid received approximately 5 to 20 μL of an algal suspension (Raphidocelis subcapitata) daily, supplemented by the addition of approximately 20 μL of GEMMA Micro 300 fish food suspension on Days 0, 1, 2 and 3. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): Gravid adults 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.
- Method of breeding: parthenogenesis.
- Source: house laboratory cultures.
- Feeding during test: fed daily
- Food type: mixture of algal suspension (Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)) and GEMMA Micro 300 fish food suspension.


ACCLIMATION

- Acclimation conditions (same as test): 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Type and amount of food: mixture of algal suspension (Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata)) and GEMMA Micro 300 fish food suspension.
- Feeding frequency: daily
- Health during acclimation (any mortality observed): No mortality

Test type:
semi-static
Water media type:
other: Reconstituted water (Elendt M4 medium)
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
not specified
Post exposure observation period:
The test organisms were observed daily throughout the test and the following recorded:
• Any mortalities (i.e. immobility), general size and condition of the parental daphnids.
• The number and general condition of the offspring produced by each parent animal.
• The number of ephippia produced.
At the end of the test, the body length and/or dry weight of the parental daphnids were observed.
Hardness:
2.5 mmol/L (= 250 mg/L as CaCO3)
Test temperature:
water temperature between 18 to 22 °C with a maximum deviation of ±1 °C during the test.
pH:
6.6 to 8.2 (control)
6.8 to 8.3 (solvent control)

Variations
1.6 (control)
1.5 (solvent control)
Dissolved oxygen:
8 mg O2/L
Salinity:
not applicable
Conductivity:
not specified
Nominal and measured concentrations:
Nominal concentrations: 0.0010, 0.032, 0.10, 0.32 and 1.0 mg/L.
Geometric mean measured test concentrations: 0.0066, 0.018, 0.046, 0.099 and 0.14 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: glass
- Type: closed
- Material, size, headspace, fill volume:
- Aeration: NO
- Type of flow-through (e.g. peristaltic or proportional diluter): Not specified
- Renewal rate of test solution (frequency/flow rate): daily
- 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
- Biomass loading rate: no data available

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water – Elendt M4 Medium:
CaCl2.2H2O 293.8 mg/L
MgSO4.7 H2O 123.3 mg/L
NaHCO3 64.8 mg/L
KCl 5.8 mg/L
Na2SiO3.9 H2O 10.0 mg/L
NaNO3 0.274 mg/L
K2HPO4 0.184 mg/L
KH2PO4 0.143 mg/L
H3BO3 2.8595 mg/L
Na2EDTA.2 H2O 2.50 mg/L
FeSO4.7 H2O 0.9955 mg/L
MnCl2.4H2O 0.3605 mg/L
LiCl 0.306 mg/L
SrCl2.6 H2O 0.152 mg/L
RbCl 0.071 mg/L
Na2MoO4.2 H2O 0.0615 mg/L
CuCl2.2 H2O 0.0165 mg/L
NaBr 0.0160 mg/L
ZnCl2 0.0130 mg/L
CoCl2.6 H2O 0.010 mg/L
KI 0.00325 mg/L
Na2SeO3 0.00219 mg/L
NH4VO3 0.000575 mg/L
Thiamine hydrochloride 0.075 μg/L
Cyanocobalamine (Vitamin B12) 0.0010 μg/L
D(+) biotin (Vitamin H) 0.00075 μg/L


OTHER TEST CONDITIONS
- Adjustment of pH: The pH was measured using a Hach Flexi handheld meter; the variation of pH for the control group was 1.6, rather than ≤ 1.5 as required by the study plan. As no significant effects on the control group were observed, this was considered not to have had an adverse impact on the study.
- Photoperiod: 16 hour light to 8 hour dark cycle was applied with a 20 minute transition period
- Light intensity: not exceed 1500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : On a daily basis the numbers of live and dead of the "Parental" (P1) generation, the numbers of live and dead "Filial" (F1) Daphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Daphnia as compared with the controls.
Filial daphnids were considered to be dead if no sign of movement was apparent during
examination. Parental Daphnia which were unable to swim for approximately 15 seconds
after gentle agitation (i.e., immobile), were considered to be dead. An immobilization
criterion for the young daphnids was considered to be inappropriate due to the large numbers
of off-spring produced in the flasks. At the end of the test, the length of each surviving parent animal was determined.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: nominal test concentrations of 0.010, 0.032, 0.10, 0.32 and
1.0 mg/L.
- Range finding study
- Test concentrations: . In the range-finding test Daphnia magna were exposed to a series of nominal concentrations of 0.10, 1.0, 10 and 100% v/v saturated solution.
- Results used to determine the conditions for the definitive study: Based on the results of the preliminary range-finding test, an initial experiment was conducted by exposing Daphnia magna (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32% v/v saturated solution for a period of 5 days. The test solutions were renewed daily throughout the 5-Day period. The experiment was terminated on Day 5 as a significant number of trapped Daphnia was observed in the 3.2, 10 and 32% v/v saturated solution test groups,
which coincided with the presence of undissolved test item in the preparations.

Further Saturated Solution Preliminary Media Preparation Trial
Given the presence of undissolved test item in the centrifuged test samples taken from the
initial experiment, a further media preparation trial was conducted using the saturated
solution method of preparation
A further experiment was conducted by exposing Daphnia magna (10 replicates of a single
daphnid per group) to solutions of the test item at nominal concentrations of 0.32, 1.0, 3.2, 10
and 32% v/v saturated solution for a period of 9 days. The concentration and stability of the test item in the test preparations were verified by chemical analysis on Days 0 and 6 (fresh media) and on Days 1 and 7 (old media). The experiment was terminated on Day 9 as chemical analysis of the freshly prepared test solutions taken on Days 0 and 6 showed that no dose-response occurred.

As visual inspection of the 3.2, 10 and 32% v/v saturated solutions prepared for use in the
initial experiment indicated the presence of undissolved test item following centrifugation,
and the analysis of the freshly prepared test solutions taken from the further initial experiment
showed that no dose-response occurred, it was considered that the use of the saturated
solution method of preparation was considered potentially inappropriate. Further media
preparation trials were therefore conducted using both a slow-stir saturated solution and
solvent spike method of preparation

Based on the results of the further media preparation trials, the use of a solvent spike method
of preparation was considered most appropriate to ensure that the maximum dissolved test
item concentration was obtained whilst minimizing the potential for the presence of
undissolved test item. A test concentration range of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L
was selected for the definitive test.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
other: EL20
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
LOEC
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
not determinable
Remarks:
Not determined
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
other: EL20
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EL10
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
morphology
Remarks:
Body Length
Duration:
21 d
Dose descriptor:
other: EL20
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
morphology
Remarks:
Body Length
Duration:
21 d
Dose descriptor:
EL50
Effect conc.:
> 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
morphology
Remarks:
Body Length
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
ca. 0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
morphology
Remarks:
Body Length
Duration:
21 d
Dose descriptor:
LOEC
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
morphology
Remarks:
Body Length
Remarks on result:
not determinable
Remarks:
Not determined
Details on results:
This study showed that there were no effects on parental immobilization or reproduction at the highest attainable test concentration (0.14 mg/L) under experimental conditions.

- Behavioural abnormalities: none
- Observations on body length and weight: After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences
(P≥0.05) between the control and the 0.018, 0.046 and 0.14 mg/L test groups; however, a
significant difference (P<0.05) was determined between the control and the 0.066 and
0.099 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test
item. As the concentrations showing a statistically significant effect did not
follow a dose response and as higher test concentrations were not significantly different from
the control, it was considered that the results obtained for the 0.0066 and 0.099 mg/L test
groups were anomalous and therefore not significant.
- Other biological observations: none
- Mortality of control:10% (control); 0% (solvent control)
- Other adverse effects control: No sub-lethal effects of exposure were observed in the solvent control or at test concentrations of 0.010, 0.10 and 1.0 mg/L; however, one Daphnia in both the control and 0.32 mg/L test group was observed to be small and also pale in colour. At 0.032 mg/L, one Daphnia was observed to be coated with algae.

- Immobilisation of control:Mortality (immobilization) occurred in the control, 0.018, 0.046, 0.099 and 0.14 mg/L test groups (10%, 10%, 10%, 50% and 10%, respectively). Statistical analysis of the mortality data by Fisher’s Exact Binomial Test with Bonferroni Correction showed that the observed mortalities at 0.018, 0.046 and 0.14 mg/L were not significantly different (P≥0.05) when compared to the control group. However, the observed mortalities in the 0.099 mg/L test group were determined to be significantly different. Given that the pattern of mortality was not dose related the effects seen on the parental daphnids were considered to be inadvertent as their occurrence cannot be attributed to any potential test item effect.
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not specified
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
N/A
Reported statistics and error estimates:
Analysis of Numbers of Live Young Produced per Adult and Analysis of the Daphnia Length Data.
Analysis of the control and solvent control group data using the Student-t test showed there was no statistically significant difference between these groups, therefore the control and solvent control test data were pooled data prior to analysis of the test concentration data.
Analysis of the number of live young produced and of the Daphnia length data from the pooled control and solvent control data and each test group were compared using the Student-t test incorporating Levene’s test on variance homogeneity and Shapiro-Wilk’s test on Normal Distribution. Mean and standard deviation values derived from the number of live young produced per surviving adult in the controls and each of the test groups were used for this analysis. No significant differences (P0.05) were found between the pooled controls and all test group in terms of the number of live young produced per adult by Day 21 using the above methods of statistical analysis.

Mean and standard deviation values derived from the length of the surviving daphnids of the parental (P1) generation in the controls and each test group were used for this analysis. No statistically significant differences (P0.05) were found between the controls and the 0.018, 0.046 and 0.14 mg/L test groups in terms of length of the surviving parental daphnids on Day 21 of the test, using the above methods of statistical analyses. There was however, a significant difference (P<0.05) determined between the control and the 0.0066 and 0.099 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item. As there was no dose response, it was considered that these statistical differences were not significant.
Validity criteria fulfilled:
yes
Conclusions:
This study showed that the test item 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) (CAS 90552-24-2) had no effects on parental immobilization, reproduction and body length at the highest attainable test concentration (0.14 mg/L) under experimental conditions.
No Observed Effect Concentration (NOEC) was determined to be 0.14 mg/L based on reproduction, immobilisation and body length.
Executive summary:

A long-term toxicity study on daphnia magna was conducted using the test item 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) (CAS 90552-24-2). The test followed the OECD TG 211 and and was GLP compliant.


Available information indicated that the test item is poorly soluble in water and therefore falls into the category of a “difficult substance” as defined by the OECD Guidance Document No. 23 (OECD, 2019). Based on the recommendations of this document and following preliminary solubility work, the use of a solvent spike method of preparation was considered most appropriate.


Due to the variability of measured test item concentrations, on the results of a preliminary range-finding test and initial experiments, it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data. The geometric mean measured test concentrations were calculated to be 0.0066, 0.018, 0.046, 0.099 and 0.14 mg/L.


Young daphnids less than 24 hours old were used for the study. Daphnids used for testing were not first brood progeny and originate from parental daphnids which were between 15-28 days old, produced first young before Day 12, have not produced ephippia 7 days prior to testing, and produced an average of three young per adult per day over the 7 days prior to the test.


The water temperature was maintained at 18 to 22 °C and a 16 hour light to 8 hour dark cycle was applied with a 20 to 30 minute transition period. The oxygen concentration remained greater than 3 mg O2/L throughout the test. Daphnids were fed daily with either an algal suspension of e.g. Raphidocelis subcapitata or a mixture of algal suspension and GEMMA Micro 300 fish food suspension.


The daphnids were observed daily throughout the test and mortalities (i.e. immobility), general size and condition of the parental daphnids, the number and general condition of the offspring produced by each parent, and the number of ephippia produced were recorded. At the end of the test, the body length and/or dry weight of the parental daphnids were observed.


Samples of the test media were taken on Day 0 and at least once per week thereafter from the definitive test for analysis. Replicate vessels were pooled to give one sample.


This study showed that there were no effects on parental immobilization or reproduction at the highest attainable test concentration (0.14 mg/L) under experimental conditions. The 21-day NOEC based on mortality (immobilization), reproduction and body length was 0.14 mg/L. 


 

Description of key information

A study determining the long-term toxicity to aquatic invertebrates (Daphnia) from the substance 2-Propenoic acid, 2-methyl-, tetracosyl ester(branched) (CAS 90552-24-2) found no effects on parental immobilization, reproduction and body length at the highest attainable test concentration (0.14 mg/L) under experimental conditions.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
> 0.14 mg/L

Additional information