2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 April 2020 - 28 August 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April 13, 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23 (2nd edition)

Version / remarks:

February 08, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Physical Description: Colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: 96.27%
Storage Conditions: At room temperature
Purity/Composition correction factor: No correction factor required
Chemical name: 2-Ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate
CAS number: 75005-95-7
EC Number: 278-051-5
Solubility in water: Insoluble
Stability in water: Not indicated

Analytical monitoring:

yes

Details on sampling:

Sampling for Analysis of Test Concentrations
Samples for possible analysis were taken from all test concentrations and the controls according to the schedule below. Stock solutions in ACN used for preparation of test solutions were sampled as well. The method of analysis is described in the appended Analytical Report (Appendix 1, appended to attached background material).

Frequency at t=0 h and t=48 h
Volume 1.0 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.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Vehicle:

yes

Remarks:

acetonitrile (ACN)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item was a colourless to pale yellow liquid with a purity of 96.27% and visually completely soluble in test medium at the concentrations tested. No correction was made for the purity/composition of the test item.
Due to the low solubility of the test item in water, test item was added directly to test medium using acetonitrile (ACN) as vehicle. Preparation of stock solutions started with the highest concentration of 1.0 mg/mL. No other treatment than vigorous shaking was needed to completely dissolve the test item in ACN (Fisher Chemical, Geel, Belgium). Lower stock concentrations, i.e. 0.010 and 0.10 mg/mL, were prepared by subsequent dilutions of the highest concentration in ACN.
Subsequently, 100 µL of each stock solution were added to respectively 1.0 L test medium to obtain the according test concentration. The mixtures were magnetically stirred for a period of 15 minutes to ensure a homogenous distribution of the test item in the test solutions. For the solvent control, 100 µL ACN were added to 1.0 L test medium. All test solutions were clear and colorless at the end of the preparation procedure.
Any residual volumes were discarded.

Test concentrations: 1.0, 10 and 100 µg/L.
Control(s): Test medium without test item or other additives (blank control) and test medium without test item but with the additive used in the treatment of the stock solutions (solvent control).

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.
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.

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.

The following salts and vitamins were added to freshly prepared test medium (see section 4.9.1.2) 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:

no

Total exposure duration:

48 h

Remarks on exposure duration:

According to OECD guideline

Hardness:

The hardness of test medium expressed as CaCO3: 180 mg/L.

Test temperature:

The temperature continuously measured in a temperature control vessel was maintained at 19°C during the test

pH:

7.8 - 8.2

Dissolved oxygen:

8.0 - 9.2 mg/L

Salinity:

Not reported: freshwater

Conductivity:

Not reported: freshwater

Nominal and measured concentrations:

Nominal concentration: 1.0, 10 and 100 µg/L
Measured concentration (0h): 0.987, 11.3 and 114 µg/L
Measured concentration (48h): 0.787, 16.7 and 61.8 µg/L

Details on test conditions:

Test type Static
Test vessels 60 mL, all-glass.
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.

Number of daphnids Controls and highest concentration: 20 per test group
Intermediate concentrations: 10 per test group
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 30 minutes after preparation of the test solutions.

Reference substance (positive control):

no

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 84 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Measured Test Item Concentrations
The results of analysis of the samples taken during the test are described in Table 2 and Table 3 of the appended Analytical Report (attached background material).
Samples taken from all stock solutions, test concentrations and the controls were analysed. The measured concentrations at the start of the test were in agreement with the nominal concentrations, i.e. were at 98-114% relative to the nominal concentrations in the stock and test solutions. During the exposure period, the concentrations in the lowest and the highest test solution decreased to 80 and 54% of initial at the end of the test, respectively. At nominally 10 µg/L, the measured concentration was at 147% relative to the initial concentration. A reason for this could not be identified.
Based on the obtained results, the average exposure concentration was calculated to be 84 µg/L in the highest test concentration and used to express effect parameters.

Immobility
Table 1 (in 'any other information on results incl. tables' below) shows the responses recorded during the test.
No immobility was observed in the controls and at any of the test concentrations throughout the exposure period.
The responses recorded in this test allowed for reliable determination of an EC50


Determination of Effect Concentrations
Table 2 (in 'any other information on results incl. tables' below) shows the effect parameters based on average exposure concentrations

Experimental Conditions
The results of measurement of pH and oxygen concentrations (mg/L) are presented in Table 4 (in 'any other information on results incl. tables' below). These test conditions remained within the limits prescribed by the study plan (pH: 6 9, not varying by more than 1.5 units; oxygen: ≥3 mg/L at the end of the test). The temperature continuously measured in a temperature control vessel was maintained at 19°C during the test and complied with the requirements as laid down in the study plan (18 - 22°C, constant within ±1°C).

Table 1 - Number of Introduced Daphnids and Incidence of Immobility in the Test

Time (h)	Replicate	Test item; Nominal conc. (µg/L)
		Blank Control	Solvent Control	1	10	100
0	A	5	5	5	5	5
	B	5	5	5	5	5
	C	5	5			5
	D	5	5			5
	Total introduced	20	20	10	10	20
24	A	0	0	0	0	0
	B	0	0	0	0	0
	C	0	0			0
	D	0	0			0
	Total immobilised	0	0	0	0	0
	Effect %	0	0	0	0	0
48	A	0	0	0	0	0
	B	0	0	0	0	0
	C	0	0			0
	D	0	0			0
	Total immobilised	0	0	0	0	0
	Effect %	0	0	0	0	0

Table 2 - Effect Parameters Based on Mean Measured Concentrations

Parameter	Test item; Nominal conc. (µg/L)
24h-, 48h-EC50	>84

Table 3 - Effect Parameters Based on Loading Rates

Parameter	Test item; Nominal conc. (µg/L)
24h-, 48h-EL50	>100

Table 4 - pH and Oxygen Concentrations (mg/L) During the Test

Test item	Start (t=0 h)		End (t=48 h)
Nominal conc. (µg /L)	O2	pH	O2	pH
Blank control	9.2	7.8	8	8.1
Solvent control	9.2	7.9	8.1	8.2
100	9.2	7.8	8.1	8.1

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, the 48h-EC50 for Daphnia magna exposed to the test item exceeded the maximum solubility of the test item in test medium, i.e. exceeded an average concentration of 84 µg/L.

The 48h-EL50 for Daphnia magna exposed to the test item exceeded the maximum solubility of the test item in test medium, i.e. exceeded a loading rate of 100 µg/L

Executive summary:

The objective of the study was to evaluate the test item 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. A combined limit/range-finding test was performed. The study met the acceptability criteria prescribed by the study plan and was considered valid. Test conditions and results of the test are presented in the table below:

Test Item:
Appearance:	Colourless to pale yellow liquid
Purity:	96.27%
Preparation of test solutions:	Nominal concentrations (µg/L), individually spiked with a stock solution in acetonitrile.
Experimental Set-up
Guideline(s):	OECD TG 202 and OECD GD 23.
Exposure design:	Static, no renewals .
Test concentration(s):	1.0, 10 and 100 µg/L.
Control(s):	Blank control and solvent control.
Stock solution(s):	0.010 – 1.0 mg/mL in acetonitrile.
Number of replicates:	Controls and highest concentration: 4 replicates containing 5 daphnids each.
	Intermediate concentrations: 2 replicates containing 5 daphnids each.
Sampling for analysis:	At t=0 and 48 hours.
Experimental conditions
pH, Temperature, Dissolved Oxygen:	Within the ranges specified in OECD TG 202.
Light regime:	A daily photoperiod of 16 hours.
Results
Actual exposure concentrations:	84 µg/L (Mean measured concentration in limit concentration)
Recorded effects:	No immobility was observed in the controls and at any of the test concentrations throughout the exposure period.

The effect parameters (based on mean measured concentrations and loading rates) obtained in this study are summarized in the table below.  

Parameter                     Test item; Nominal conc. (µg /L)

24h-, 48h-EC50              >84

 

Parameter                     Test item; Loading rate (µg/L)

24h-, 48h-EL50              >100

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

84 µg/L

Additional information

The 48h-EC50 for Daphnia magna exposed to the test item exceeded the maximum solubility of the test item in test medium, i.e. exceeded an average concentration of 84 µg/L.

The 48h-EL50 for Daphnia magna exposed to the test item exceeded the maximum solubility of the test item in test medium, i.e. exceeded a loading rate of 100 µg/L