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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 May 2019 - 16 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
Adopted April 13, 2004
Deviations:
no
Qualifier:
according to
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
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: Esacure 3644
Physical Description: Light yellow solid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below. In addition, the filter containing the undissolved residue was kept for possible analysis . The method of analysis is described in the appended Analytical Report (Appendix 2).

Frequency at t=0 h and t=48 h
Volume 2.0 mL from the approximate centre of the test vessels
Storage Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling. All solutions containing the test item were protected from light.

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.
Vehicle:
no
Details on test solutions:
The batch of Esacure 3644 tested was a light yellow solid UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions was performed under dimmed light conditions and glassware used was wrapped in aluminium foil to minimize exposure to light.

Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.

Any residual volumes were discarded.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
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.
Reason for selection: This system has been selected as an internationally accepted invertebrate species.
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.

ENVIRONMENT
Temperature of medium: 18 to 22°C
Feeding: Daily, a suspension of fresh water algae.
Culture medium: M7, as prescribed by Dr. Elendt-Schneider (Elendt, B.-P., 1990: Selenium deficiency in Crustacea. An ultrastructural approach to antennal damage in Daphnia magna Straus. Protoplasma 154, 25-33).

The following salts and vitamins were added to freshly prepared test medium to reach the following concentrations:

Salts: H3BO3 0.71 mg/L
FeSO4.7H2O 0.25 mg/L
MnCl2.4H2O 0.090 mg/L
LiCl 0.076 mg/L
RbCl 0.018 mg/L
SrCl2.6H2O 0.038 mg/L
Na2MoO4.2H2O 0.015 mg/L
NaBr 0.0040 mg/L
CuCl2.2H2O 0.0042 mg/L
ZnCl2 0.013 mg/L
CoCl2.6H2O 0.010 mg/L
KI 0.0032 mg/L
Na2SeO3 0.0022 mg/L
NH4VO3 0.00057 mg/L
Na2EDTA.2H2O 0.62 mg/L
Na2SiO3.5H2O 7.5 mg/L
NaNO3 0.27 mg/L
KH2PO4 0.14 mg/L
K2HPO4 0.18 mg/L

Vitamins: Thiamine hydrochloride 75.0 µg/L
B12 1.0 µg/L
Biotin 0.75 µg/L
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
N/A
Hardness:
The hardness of test medium expressed as CaCO3: 180 mg/L
Test temperature:
19 to 20 °C
pH:
7.8 to 8.2
Dissolved oxygen:
8.6 to 9.2 mg/L
Salinity:
Not measured
Conductivity:
Not measured
Nominal and measured concentrations:
Loading rate Concentration analyzed
[mg/L] [mg/L]
0 <0.0008
1.0 <0.0008
10 <0.0008
100 0.00629
0 <0.0008
1.0 <0.0008
10 <0.0008
100 0.00434
Details on test conditions:
Test vessels 60 mL, all-glass.
Test type Static
Aeration No aeration of the test solutions was applied.

Number of daphnids Control and highest concentration: 20 per test group
Intermediate concentrations: 10 per concentration
Loading 5 per vessel containing 50 mL of test solution.
Introduction of daphnids Within 50 minutes after preparation of the test solutions.

Test duration 48 hours
Light The study was performed in the dark.
Feeding No feeding
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 5.2 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mobility
Remarks on result:
other: Loading rate
Details on results:
Table 1 shows the responses recorded during the test. No immobility was observed in the control and at any of the test concentrations throughout the exposure period.
The 48h-EC50 for Daphnia magna exposed to Esacure 3644 exceeded the maximum solubility of the test item in test medium at a loading rate of 100 mg/L, i.e. exceeded an average exposure concentration of 5.2 µg/L.
The 48h-EL50 for Daphnia magna exposed to Esacure 3644 was beyond the range tested, i.e. exceeded a loading rate of 100 mg/L.
Results with reference substance (positive control):
The 24h-EC50 was 0.92 mg/L with a 95% confidence interval between 0.78 and 1.1 mg/L.
The 48h-EC50 was 0.55 mg/L with a 95% confidence interval between 0.47 and 0.64 mg/L.

TABLE 1 - Number of introduced Daphnids and Incidence of immobility

Time (h) Replicate Esacure 3644; Loading rate (mg/L)
Control 1 10 100
0 A 5 5 5 5
B 5 5 5 5
C 5 5
D 5 5
Total introduced 20 10 10 20
24 A 0 0 0 0
B 0 0 0 0
C 0 0
D 0 0
Total immobilised 0 0 0 0
Effect % 0 0 0 0
48 A 0 0 0 0
B 0 0 0 0
C 0 0
D 0 0
Total immobilised 0 0 0 0
Effect % 0 0 0 0

TABLE 2 - POSITIVE CONTROL

K2Cr2O7 Number Exposed % immobile1  
Nominal conc. (mg/L)   t=24h                      t=48h
Control 20 0 0
0.1 20 0 0
0.18 20 5 5
0.32 20 5 5
0.56 20 0 50
1 20 60 100
1.8 20 95 95

INTERPRETATION

Acceptability of the Test:

1.       In the control, no daphnids became immobilised or showed other signs of disease or stress such as discoloration or unusual behaviour 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.

Data Handling:

All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

The average exposure concentrations were calculated as  , being the geometric means of the concentrations of Esacure 3644 measured in the samples taken at the start (Ct=0) and the end of the test (Ct=48).

Analysis:

No EC50 could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested).

No EL50 could be calculated because the test item proved to be non-toxic (EL50 > maximum loading rate tested).

Computerized systems:

Critical computerized systems used in the study are listed below or presented in the appropriate Phase Report.  All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

System name Version No. Description of Data Collected and/or Analyzed
Deviation Information Library 2.1 Deviations
REES Centron SQL 2.0 Temperature, relative humidity and/or
atmospheric pressure monitoring
Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the 48h-EC50 for Daphnia magna exposed to Esacure 3644 exceeded the maximum solubility of the test item in test medium at a loading rate of 100 mg/L, i.e. exceeded an average exposure concentration of 5.2 µg/L.
In conclusion, the 48h-EL50 for Daphnia magna exposed to Esacure 3644 was beyond the range tested, i.e. exceeded a loading rate of 100 mg/L.
Executive summary:

The objective of the study was to evaluate Esacure 3644 for its ability to generate acute toxic effects on the mobility of Daphnia magna during an exposure period of 48 hours and, if possible, to determine the EC50 at 24 and 48 hours of exposure.

The study procedures described in this report were based on the OECD guideline No. 202, 2004. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2019.

The batch of Esacure 3644 tested was a light yellow solid UVCB and not completely soluble in test medium at the loading rates initially prepared.

Water Soluble Fractions (WSFs) were individually prepared at loading rates ranging between 1.0 and 100 mg/L and used as test concentrations. Preparation of test solutions was performed under dimmed light conditions and glassware used was wrapped in aluminium foil to minimize exposure to light.

A combined limit/range-finding test was performed. Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to a WSF prepared at a loading rate of 100 mg/L, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to WSFs individually prepared at loading rates of 1.0 and 10 mg/L in the combined range-finding test. The total exposure period was 48 hours and samples for analytical confirmation of exposure concentrations were taken at the start and at the end of the test. Since the test item was expected to be sensitive to light, the exposure was performed in the dark.

No immobility was observed in the control and at any of the test concentrations throughout the exposure period.

In the limit concentration, 6.3 and 4.3 µg/L were measured at the start and end of the test, respectively. Accordingly, the average exposure concentration was calculated to be 5.2 µg/L.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

In conclusion, the 48h-EC50 for Daphnia magna exposed to Esacure 3644 exceeded the maximum solubility of the test item in test medium at a loading rate of 100 mg/L, i.e. exceeded an average exposure concentration of 5.2 µg/L. The 48h-EL50 for Daphnia magna exposed to Esacure 3644 was beyond the range tested, i.e. exceeded a loading rate of 100 mg/L.

Description of key information

Study performed to recognised OECD guidlines with GLP certification

Key value for chemical safety assessment

Additional information

The 48h-EC50 for Daphnia magna exposed to Esacure 3644 exceeded the maximum solubility of the test item in test medium at a loading rate of 100 mg/L, i.e. exceeded an average exposure concentration of 5.2 µg/L