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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:
From November 2017 to April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from all test concentrations and the control at t=0h and t=48h.
- Sampling method: 2.0 mL from the approximate centre of the test vessels. At the end of the exposure period, the replicates were pooled at each concentration before sampling
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility
Vehicle:
no
Details on test solutions:
PREPARATION OF TEST SOLUTION:
The batch of 1,3-Bis (4-hydroxy benzoyl) benzene tested was an off white crystalline powder with a purity of 99.53% and not completely soluble in test medium at the loading rate initially prepared. No correction was made for the purity/composition of the test item.
Preparation of test solutions started with a loading rate of 100 mg/L applying a 15-minute period of ultrasonic waves followed by a one-hour period of magnetic stirring to ensure maximum dissolution of the test item in test 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.
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: For the test selection of young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old

BREEDING
- Start of each batch: With newborn daphnids, i.e. less than 3 days old, by placing about 250 of them 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.
- Medium :M7, as prescribed by Dr. Elendt-Schneider


Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Hardness:
The hardness of the test medium : 180 mg/L expressed as CaCO3
Test temperature:
between 20 and 21°C during the test
pH:
between 8.0 and 8.7 during the test
Dissolved oxygen:
between 8.5 and 9.5 mg/L during the test
Salinity:
NA
Nominal and measured concentrations:
Solutions containing 1.0, 10, and 100% of a Saturated Solution (SS) prepared at a loading rate of 100 mg/L.
(Range Finding test: Solutions containing 1.0, 10 % of a SS prepared at a loading rate of 100 mg/L and Limit test: 100 % of a SS prepared at a loading rate of 100 mg/L).
Details on test conditions:
TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Type (delete if not applicable): open
- Aeration: No aeration of the test solutions.
- No. of organisms per vessel: 5 per vessel containing 50 mL of test solution
- No. of vessels per concentration (replicates): 20 each in the control and the highest test concentration, 10 per intermediate test concentration.
- Feeding : No feeding
- Introduction of daphnids : Within 19 minutes after preparation of the test solutions.

TEST MEDIUM / WATER PARAMETERS
- Medium: Adjusted ISO medium

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 hours photoperiod daily


EFFECT PARAMETERS MEASURED:
- Immobility (including mortality): At 24 hours and at 48 hours.
- pH and dissolved oxygen: At the beginning and at the end of the test, for the highest test concentration and the control.
- Temperature of medium: continuously in a temperature control vessel, beginning at the start of the test.

TEST CONCENTRATIONS:
- Range Finding test: Solutions containing 1.0, 10 % of a SS prepared at a loading rate of 100 mg/L.
- Limit test: 100 % of a SS prepared at a loading rate of 100 mg/L.
- Results used to determine the conditions for the definitive study: not needed
Reference substance (positive control):
yes
Remarks:
K2Cr2O7
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 0.33 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LL50
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.33 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Measured Concentrations:
Samples taken from the highest test concentration were analysed. At the start of the test a concentration of 0.33 mg/L was measured in the undiluted SS. During the exposure period, this concentration remained constant, i.e. it was 94% of initially measured at the end of the test.
Based on these results, effect parameters were based on measured concentrations.

- Immobility:
No immobility was observed in the control or at any of the concentrations tested throughout the exposure period.
Results with reference substance (positive control):
The actual responses in this reference test with K2Cr2O7 are within the ranges of the expected responses at the different concentrations, i.e. the 48h-EC50 was between 0.3 and 1.0 mg/L.
The 24h-EC50 was 0.59 mg/L with a 95% confidence interval between 0.52 and 0.68 mg/L.
The 48h-EC50 was 0.41 mg/L with a 95% confidence interval between 0.34 and 0.46 mg/L.

Reported statistics and error estimates:
No EC50 could be calculated because the test item proved to be non-toxic (EC50 > maximum soluble concentration tested)
Validity criteria fulfilled:
yes
Conclusions:
The 48h-EC50 for Daphnia magna exposed to 1,3-Bis (4-hydroxy benzoyl) benzene was >0.33 mg/L, being considered to be the maximum soluble concentration of the test item in test medium at a loading rate of 100 mg/L.
Executive summary:

The objective of the study was to evaluate 1,3-Bis (4-hydroxy benzoyl) benzene 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 was compliant with the OECD guideline No. 202, 2004. In addition, as the 1,3-Bis (4-hydroxy benzoyl) benzene was poorly soluble in the test medium, procedures were also based on the test methods described in the OECD series on testing and assessment number 23, 2000.

 

Thus, a Saturated Solution (SS) was prepared at a loading rate of 100 mg/L and used as the highest concentration. Lower concentrations were prepared by diluting the highest concentration in test medium.

A combined limit/range-finding test was performed.Twenty daphnids per group (5 per replicate, quadruplicate) were exposed to an untreated control and to the undiluted SS, in a limit test. In addition ten daphnids per group (5 per replicate, duplicate) were exposed to 1.0 and 10% of the SS 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.

 

No biological relevant immobility was observed in the control and at any test concentrations throughout the test.

Samples taken from the undiluted SS were analysed. At the start of the test, the actual exposure concentration was 0.33 mg/L, which remained stable during the test, i.e. it was 94% of initially measured at the end of the test. Based on these results, effect parameters were expressed in terms of the initial measured concentrations (mg/L). 

 

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

 

In conclusion, under the conditions of the present study, no immobility was observed at any of the concentrations of 1,3-Bis (4-hydroxy benzoyl) benzene tested.

The 48h-EC50 for Daphnia magna exposed to 1,3-Bis (4-hydroxy benzoyl) benzene was > 0.33 mg/L, being considered to be the maximum soluble concentration of the test item in test medium a loading rate of 100 mg/L. The 48h-NOEC was ≥ 0.33 mg/L.

Description of key information

The toxicity of the 1,3-Bis (4-hydroxy benzoyl) benzene to the freshwater flea Daphnia magna was investigated in a GLP-compliant study performed in accordance with OECD Guideline No. 202. In addition, due to the low solubility of the test item, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2000.

Under the conditions of the study, no immobility was observed at any of the concentrations of 1,3-Bis (4-hydroxy benzoyl) benzene tested. The EC50 for immobilization (48h-EC50) for Daphnia magna was > 0.33 mg/L, measured concentration being considered to be the maximum soluble concentration of the test item in test medium at a loading rate of 100 mg/L.

Key value for chemical safety assessment

Additional information

The toxicity of the test item to freshwater flea Daphnia magna was investigated in one GLP-compliant study performed in accordance with standard methods, without deviations. The study is considered as reliable (Klimisch 1) and is selected as a key study for the endpoint.