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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 July 2020 - 03 September 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Guidance document on aqueous-phase Aquatic Toxicity Testing of Difficult Test Chemicals, OECD series on testing and assessment number 23
Version / remarks:
2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
adopted 13 April 2014
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione
Cas Number:
54024-17-8
Molecular formula:
C20 H26 O2
IUPAC Name:
(3aS,3bS,9aR,9bS,11aS)‐11a‐ethyl‐10‐methylidene‐1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH‐cyclopenta[a]phenanthrene‐1,7‐dione
Test material form:
solid: particulate/powder
Details on test material:
Storage Conditions: In refrigerator (2-8°C)
Specific details on test material used for the study:
Physical Description: Off-white powder
Storage conditions: In refrigerator (2-8°C)
Test item handling: No specific handling conditions required

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
- Frequency: at t=0 h and t=48 h
- Volume: 2.0 mL from the approximate centre of the test vessels.
- Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.
Additionally, reserve samples of 2.0 mL were taken for possible analysis. If not used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Vehicle:
no
Details on test solutions:
Preparation of test solutions started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Saturated Solution (SS) was collected by filtration through a 0.45 μm membrane filter (RC55, Whatman) and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium. All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
- Species: Daphnia magna (Crustacea, Cladocera) (Straus, 1820), at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Source: In-house laboratory culture with a known history.
- Validity of batch: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Characteristics: Daphnia, less than 24 hours old, from parental daphnids of more than two weeks old

BREEDING
- Start of each batch: Approximately 250 newborn daphnids, i.e. less than 3 days old, were placed into 5 litres of medium in an all glass culture vessel.
- Maximum age of the cultures: 4 weeks
- Renewal of the cultures: After 7 days of cultivation, half of the medium twice a week.
- Temperature of medium: 18-22°C
- Feeding: Daily, a suspension of fresh water algae.
- Culture medium: M7, as described by OECD 211 TG.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
The hardness of test medium expressed as CaCO3: 180 mg/L
Test temperature:
between 19 and 20 °C
pH:
Start of the test: 7.8;
End of the test: 7.9 - 8.0
Dissolved oxygen:
Start of the test : 8.9;
End of the test : 9.0 - 9.1
Nominal and measured concentrations:
Nominal concentrations: Solutions containing 10, 18, 32, 56 and 100% of the SS, prepared at a loading rate of 100 mg/L.
Measured concentrations: The average exposure concentrations were 3.8, 6.7, 11, 22 an 39 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessels: 60 mL, all-glass
- Number of daphnids: 20 per concentration
- Loading: 5 per vessel containing 50 mL of test solution.
- Light: A daily photoperiod of 16 hours.
- Feeding: No feeding
- Aeration: No aeration of the test solutions was applied.
- Introduction of daphnids: Within 20 minutes after preparation of the test solutions.

TEST MEDIUM
The following salts (analytical grade) were added to tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):
CaCl2.2H2O: 211.5 mg/L
MgSO4.7H2O: 88.8 mg/L
NaHCO3 46.7: mg/L
KCl: 4.2 mg/L
The hardness of test medium expressed as CaCO3: 180 mg/L

EFFECT PARAMETERS MEASURED:
Immobility (including mortality): At 24 hours and at 48 hours.

SATURATION TEST
A saturation test, intended to determine the time of mixing required to reach the maximum soluble concentration was performed. A loading rate of 100 mg/L was prepared and continuously stirred for three days.
Approximately every 24 hours, subsamples of approximately 10 mL were collected by
filtration through a 0.45 μm syringe membrane filter (RC-S, Whatman) before the sample for
chemical analysis was taken.
The concentrations measured in the solution prepared at a loading rate of 100 mg/L were 34 mg/L, 38 and 40 mg/L after 24, 48 and 72 hours of magnetic stirring, respectively.

COMBINED LIMIT RANGE -FINDING TEST
Ten daphnids per concentration (in duplicate, 5 per vessel) were exposed to solutions containing 0.10, 1.0 and 10% of the SS prepared at 100 mg/L in the combined range-finding test. Twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and a SS prepared at a loading rate of 100 mg/L,
No immobility was observed in the control and at the three lowest test concentrations
throughout the exposure period, while 35 and 85% of the daphnids were immobilized in the
highest test group after 24 and 48 hours of exposure, respectively. The results were used to determine the conditions for the definitive study.

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
16 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 14- 19 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
20 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Remarks on result:
other: 95% CI: 18-23 mg/L
Details on results:
Analytical results
The measured concentrations at the start of the test were 3.9, 6.5, 12, 22 and 39 mg/L in solutions containing 10, 18, 32, 56 and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. The concentrations remained stable during the 48-hour exposure period, i.e. were at 93-104% relative to the initial concentrations at the end of the test. No test item was detected in the control sample. Based on these results, the average exposure concentrations were calculated and used to express effect parameters.

Biological results: For detail see table 1 in "any other information on results".
No immobility was recorded in the control group and at the concentration of 6.7 mg/L, while a single daphnid (i.e. 5%) did not survive exposure for 48-hours to the lowest concentration tested.
A dose-related increase of immobility was observed at concentrations of 11 mg/L and higher, resulting in 100% immobility at the
highest test concentration.

Validity of the Test
1. In the control, not more than 10% of the daphnids became immobilised (i.e. 0%) or showed other signs of disease or stress (i.e. 5%), for example discoloration or unusual behavior such as trapping at the surface of the medium.
2. The dissolved oxygen concentration at the end of the test was ≥3 mg/L in control and test vessels.

Any other information on results incl. tables

 


Table: Measured Concentrations (mg/L)










































EMETAM %SS prep. at a loading rate of 100 mg/L



Measured concentration (mg/L)


T=0h



Measured concentration (mg/L)


T=48h


Average exposure concentration (mg/L)
103.93.63.8
186.56.86.7
32121111
56222222
100393939

Table : Effects observed in the final test.

























































































































































































TimeReplicateControl3.8 mg/L6.7 mg/L11 mg/L22 mg/L39 mg/L
0A555555
 B555555
 C555555
 D555555
 Total introduced202020202020
24A000025
 B00(2)004 (5)5 (5)
 C0 (1)00035
 D00004#5
 

Total immobilised


00001320
 Effect %000065100
48A010125
 B000155
 C000155
 D000145
 Total immobilised01041620
 Effect %0502080100

(   ) Number of daphnis observed trapped at the surface of the test solution. These organisms were reimmersed into the respective solution before recording of immobility.


# Microscopic observation revealed no test item attached to the daphnids.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For details: see "details on results".
Conclusions:
In conclusion, the 48h-EC50 for Daphnia magna exposed to EMETAM was 16 mg/L based on
average exposure concentrations (95% confidence interval between 14 and 19 mg/L).
Executive summary:

Short-term toxicity to aquatic invertebrates Daphnia magna was assessed according to OECD guideline 202 and in compliance with GLP.  Twenty Daphnids per group (5 per replicate, quadruplicate) were exposed under static conditions to 10, 18, 32, 56, and 100% of the saturated solution prepared at a loading rate of 100 mg/L in fresh water.


The measured concentrations at the start of the test were 3.9, 6.5, 12, 22, and 39 mg/L in solutions containing 10, 18, 32, 56, and 100% of the SS prepared at a loading rate of 100 mg/L, respectively. The concentrations remained stable during the 48-hour exposure period, i.e. were at 93-104% relative to the initial concentrations at the end of the test. The average exposure concentration was 3.8, 6.7, 11, 22, and 39 mg/L.


A dose-related increase of immobility was observed at concentrations of 11 mg/L and higher, resulting in 100% immobility at the highest test concentration.


The 48h-EC50 for Daphnia magna exposed to EMETAM was 16 mg/L based on average exposure concentrations (95% confidence interval between 14 and 19 mg/L).


All the validity criteria were met and therefore the study is considered to be valid.