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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:
September 17, 2019 to November 21, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
Due to the low aqueous solubility and complex nature of the test substance, for the test, the test medium was prepared as water accommodated fractions (WAFs).
Details on test solutions:
The reconstituted water (Elendt M4 medium) used for the two range-finding and one definitive test was the same as that used to maintain the stock animals. For detail composition please refer attached background material.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: In-house laboratory cultures
- Feeding during test
- Food type: a mixture of algal suspension (Raphidocelis subcapitata) and GEMMA Micro 300 fish food suspension
- Frequency: daily

Adult Daphnia were maintained in 150 mL glass vessels containing Elendt M4 medium in a temperature-controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 h light and 8 h darkness cycle with 20-minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata (formally known as Pseudokirchneriella subcapitata)) and GEMMA Micro 300 fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. 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. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

EXPOSURE CONDITIONS:
For each concentration, a single daphnid was placed in 100 mL of the test preparation in 120 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 maximum deviation of ±1 °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 control group was maintained under identical conditions but not exposed to the test substance. The test preparations were renewed daily throughout the test. The adult Daphnia were transferred to fresh media 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 before being discarded. Each daphnid received approximately 5 to 20 μL of an algal suspension (Raphidocelis subcapitata) daily and approximately 20 μL of GEMMA Micro 300 fish food suspension on Days 0 to 3 only. 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 type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
240 to 280 mg/L as CaCO3. The water hardness of the control and the highest surviving test concentration in the fresh and old media was measured once per week. Water hardness was determined using the method described in fields on-site methods for analysis of water (British Standards Institution, 1993).
Test temperature:
18 - 22°C
pH:
7.9 ±0.3
Dissolved oxygen:
Please refer to the attached background material
Nominal and measured concentrations:
First range finding test: 10 and 100 mg/L (nominal)
Second range finding test: 0.010, 0.10, 1.0 and 10 mg/L
Definitive test: 2.6, 6.4, 16, 40 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 120 mL glass vessel
- Type (delete if not applicable): closed
- Aeration: Not specified
- Renewal rate of test solution (frequency/flow rate): The test preparations were renewed on Days 0, 2, 5, 7 and 9.
- No. of vessels per concentration (replicates): 10 replicates of a single daphnid per group

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light (not exceeding 1500 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 1500 Lux

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: first range finding test: 10 and 100 mg/L (was terminated on Day 5 due to 60% mortality) and for second range finding test: 0.010, 0.10, 1.0 and 10 mg/L.
- Results used to determine the conditions for the definitive study: 60% immobilization was observed at 100 mg/L and 40% immobilization was observed at 10 mg/L. Sub-lethal effects of exposure were observed at test concentrations of 10 mg/L and above. These responses were small and pale in color. In the second range-finding test no immobilization or sub-lethal effects were observed in the control, 0.010, 0.10, 1.0 and 10 mg/L test groups. Chemical analysis of the fresh test preparations on Day 0 showed measured concentrations to range from less than the limit of quantification (LOQ) to 0.40 mg/L. The chemical analysis of the old/expired test preparations on Day 2 showed measured concentrations to range from less than the LOQ to 0.019 mg/L indicating that the test item was unstable under test conditions. Based on this findings the definitive test concentrations were selected.

Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
ca. 100 other: mg/L LR WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
ca. 100 other: mg/L LR WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
ca. 6.4 other: mg/L LR WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
ca. 16 other: mg/L LR WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOELR
Effect conc.:
ca. 2.6 other: mg/L LR WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Body length
Key result
Duration:
21 d
Dose descriptor:
LOELR
Effect conc.:
ca. 6.4 other: mg/L LR WAF
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Body length
Details on results:
- Behavioural abnormalities: These sub-lethal effects were all surviving daphnids were observed to be small on Days 6 and 7, and on Day 8 four of the 10 daphnids were observed to pale in color compared to the controls, and on Days 9 and 10 six of the 10 daphnids were observed to be pale in color compared to the controls. From Day 11 onwards all surviving daphnids were observed to be normal.
- Observations on body length: The results showed that there were no statistically significant differences (P≥0.05) between the control and the 2.6 mg/L test group in terms of length of the daphnids after 21 days exposure to the test item. The 6.4, 16, 40 and 100 mg/L test groups showed a statistically significant difference from the control after 21 days in terms of length of the daphnids.
- Mortality of control: No mortalities occurred in the 2.6, 6.4, 16, 40 and 100 mg/L loading rate WAF groups throughout the test. However one mortality occurred in the control on Day 17.
- Immobilisation of control: In the initial range-finding test no immobilization was observed in the control.


Reported statistics and error estimates:
The 21-Day values for parental generation immobilization were calculated by Probit analysis using Linear Maximum-Likelihood Regression. The values for reproduction after 21 days were calculated using Linear analysis using Linear Maximum-Likelihood Regression. The values for body length were calculated using the 3-parameter normal cumulative distribution function. All determinations were performed using the ToxRatPro software package (TOXRAT).

Summary of results

 

Endpoint

Concentration (mg/L LR WAF)

95% Confidence Limits (mg/L LR WAF)

 

 

 

Immobilization

EL10

>100

Not determined*

EL20

>100

Not determined*

EL50

>100

Not determined*

No Observed Effect Loading Rate

100

-

Lowest Observed Effect Loading Rate

Not determined

-

 

 

 

Reproduction

EL10

11

6.8 – 14

EL20

26

20 – 31

EL50

>100

Not determined*

No Observed Effect Loading Rate

6.4

-

Lowest Observed Effect Loading Rate

16

-

 

 

 

Body Length

EL10

38

25 – 57

EL20

>100

Not determined*

EL50

>100

Not determined*

No Observed Effect Loading Rate

2.6

-

Lowest Observed Effect Loading Rate

6.4

-

 

 

Please refer to the background materials for tables.

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the NOELR for effects on immobilisation (mortality), body length and reproduction were determined to be 100, 2.6 and 6.4 mg/L LR WAF respectively.
Executive summary:

A study was conducted to determine the long-term toxicity of the test substance 'mono- and di- C16-18 PSE and C16-18 AE10 PSE' to Daphnia magna according to OECD Guideline 211, in compliance with GLP. Due to the low aqueous solubility and complex nature of the test substance, the test medium was prepared as water accommodated fractions (WAFs). Based on the results of preliminary range-finding tests, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test substance over a range of nominal loading rates of 2.6, 6.4, 16, 40 and 100 mg/L for a period of 21 days. The test solutions were renewed daily throughout the test. The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with an algal suspension and with a mixture of algal suspension and GEMMA Micro 300 fish food suspension on Days 0 to 3. Chemical analysis of the fresh test preparations on Days 0, 6, 13, and 20 showed measured test concentrations to range from 0.012 to 3.7 mg/L. Chemical analysis of the aged test preparations on Days 1, 7, 14, and 21 showed measured test concentrations to range from less than the limit of detection, which was determined to be 0.0040 to 3.2 mg/L. 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. No mortalities occurred in the 2.6, 6.4, 16, 40, and 100 mg/L loading rate (LR) WAF groups throughout the test. Statistically significant differences in body weight length were observed in 6.4, 16, 40, and 100 mg/L LR WAF test groups. After 21 days, effects on reproduction in terms of producing a fewer number of live young per adult was observed in 16, 40, and 100 mg/L LR WAF test groups. Information on the effects of the test substance on the filial (F1) generation is limited since the young were removed soon after liberation from the brood pouch. Seven of the broods produced on Day 11 in the 100 mg LR WAF group, one of the broods produced on Day, 14 in the 40 mg/L LR WAF group and two of the broods produced on Day 15 in the 40 and 100 mg/L LR WAF test groups, were observed to be pale. Young was first produced in the control test group on Day 7 and 8 from nine of the parental Daphnia and one of the parental Daphnia had the first brood on Day 10 of the test. Under the conditions of the study, the NOELR for effects on immobilisation (mortality), body length, and reproduction were determined to be 100, 2.6, and 6.4 mg/L LR WAF respectively (Covance, 2019).

Description of key information

Based on the results of the study, the NOELR for effects on immobilisation (mortality), body length, and reproduction were determined to be 100, 2.6, and 6.4 mg/L LR WAF (nominal) respectively.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.6 mg/L

Additional information

A study was conducted to determine the long-term toxicity of the test substance 'mono- and di- C16-18 PSE and C16-18 AE10 PSE' to Daphnia magna according to OECD Guideline 211, in compliance with GLP. Due to the low aqueous solubility and complex nature of the test substance, the test medium was prepared as water accommodated fractions (WAFs). Based on the results of preliminary range-finding tests, Daphnia magna were exposed (10 replicates of a single daphnid per group) to a WAF of the test substance over a range of nominal loading rates of 2.6, 6.4, 16, 40 and 100 mg/L for a period of 21 days. The test solutions were renewed daily throughout the test. The numbers of live and dead adultDaphniaand young daphnids (live and dead) were determined daily. TheDaphniawere fed daily with an algal suspension and with a mixture of algal suspension and GEMMA Micro 300 fish food suspension on Days 0 to 3. Chemical analysis of the fresh test preparations on Days 0, 6, 13, and 20 showed measured test concentrations to range from 0.012 to 3.7 mg/L. Chemical analysis of the aged test preparations on Days 1, 7, 14, and 21 showed measured test concentrations to range from less than the limit of detection, which was determined to be 0.0040 to 3.2 mg/L. 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. No mortalities occurred in the 2.6, 6.4, 16, 40, and 100 mg/L loading rate (LR) WAF groups throughout the test. Statistically significant differences in body weight length were observed in 6.4, 16, 40, and 100 mg/L LR WAF test groups. After 21 days, effects on reproduction in terms of producing a fewer number of live young per adult was observed in 16, 40, and 100 mg/L LR WAF test groups. Information on the effects of the test substance on the filial (F1) generation is limited since the young were removed soon after liberation from the brood pouch. Seven of the broods produced on Day 11 in the 100 mg LR WAF group, one of the broods produced on Day, 14 in the 40 mg/L LR WAF group and two of the broods produced on Day 15 in the 40 and 100 mg/L LR WAF test groups, were observed to be pale. Young was first produced in the control test group on Day 7 and 8 from nine of the parental Daphnia and one of the parentalDaphniahad the first brood on Day 10 of the test. Under the conditions of the study, the NOELR for effects on immobilisation (mortality), body length, and reproduction were determined to be 100, 2.6, and 6.4 mg/L LR WAF (nominal) respectively (Covance, 2019).