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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

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:

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 4, attached background material).

Frequency at t=0 h and t=72 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.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the highest test concentration but without algae and samples for analysis were taken at the start and at the end of the test period.

Vehicle:

yes

Remarks:

acetonitrile (ACN)

Details on test solutions:

Preparation of Test Solutions
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.

After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.
Any residual volumes were discarded.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST SYSTEM
Species Raphidocelis subcapitata, strain: NIVA CHL 1
Source In-house laboratory culture.
Reason for selection This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

According to OECD guideline

Hardness:

(Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

22 - 23 °C

pH:

7.9 - 8.1

Dissolved oxygen:

Not reported

Salinity:

Not reported - freshwater

Conductivity:

Not reported - freshwater

Nominal and measured concentrations:

Nominal: 1.0, 10 and 100 µg/L
Measured (0h): 0.786, 10.0 and 105 µg/L
Measured (72h): 0.34, 3.14 and 30.8 µg/L

Details on test conditions:

FRESHWATER ALGAE CULTURE
Stock culture
Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.

Light intensity 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture:
Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

Pre-culture medium M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

CONTROLS AND REPLICATES
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).
Replicates 6 replicates of each control and the highest test concentration,
3 replicates of each intermediate test concentration,
1 replicate of each solvent-treated test group without algae.

TEST PROCEDURE AND CONDITIONS
Test duration 72 hours
Test type Static
Test vessels 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
Test Medium M2 (see paragraph 4.8.)
Cell density An initial cell density of 1 x 104 cells/mL.
Illumination Continuously using TLD-lamps with a light intensity within the range of 86 to 87 µE.m-2.s-1.
Incubation Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENTS AND RECORDINGS
pH At the beginning and at the end of the test, for the limit concentration and the controls.
Temperature of medium Continuously in a temperature control vessel.
Appearance of the cells At the end of the test, microscopic observations were performed on the limit concentration and the controls to observe for any abnormal appearance of the algae.

Recording of Cell Densities
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

57 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

57 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 57 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 57 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 57 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 57 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 µg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Measured Test Item Concentrations
The results of analysis of the samples taken during the combined limit/range-finding test are described in Table 2 and Table 3 of the appended Analytical Report (apppended to 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 79-105% relative to the nominal concentrations in the stock and test solutions. During the exposure period, the concentrations in the test solutions decreased to 29-44% of initial at the end of the test.

Small responses were detected in the samples taken from the controls, which were, most likely related to contamination during sampling. Considering the low amounts detected and that algal growth was found to be higher in the test item treated groups, this was considered to be negligible.
The concentrations measured in the samples taken from solutions with algae were comparable with the concentrations measured in the samples without algae at the start of the test. At the end of the test, the concentration in the sample without algae was clearly lower than in the sample with algae, i.e. was at 3.1% relative to the initial concentration, indicating that the presence of the algae affected the concentration of the test item in test medium throughout the test.

Based on the obtained results, the mean measured exposure concentration was calculated to be 57 µg/L in the highest test concentration and used to express effect parameters.


Mean Cell Densities
Figure 1 (attached below) shows growth curves at in the controls and the limit concentration of the test item. The individual and group mean cell densities measured at 24h intervals are given in Table 6 (appendix 1, appended to attached background material).

Inhibition of Growth Rate and Inhibition of Yield
Table 1 (below) shows group mean growth rates and the percentages of growth rate inhibition (total test period), whereas Table 2 (below) shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 3 below (see Appendix 1 for the individual values, appended to background material). Statistical analysis of the data is shown in Appendix 2 and Appendix 3, appended to background material.
At the end of the test, cell densities in both control groups were below the cell densities measured for the test item exposed groups, indicating a stimulation rather than an inhibition of algal growth due to test item exposure.

Statistical comparison of algal growth between the blank and solvent control indicated that a significant difference was present for both growth rate and yield (see Appendix 2 and Appendix 3, appended to attached background material). Recorded data from the two control groups were therefore not pooled and only the solvent control was used as a reference to determine growth rate and yield inhibition at the different test item concentrations.

No statistically significant inhibition was recorded for growth rate or yield at any of the concentrations tested when compared to the solvent control group.
Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest test concentration when compared to the control.

Determination of Effect Concentrations
Table 4 (below) shows the effect parameters based on mean measured exposure concentrations.
Table 5 (below) shows the effect parameters based on loading rates.

Experimental Conditions
Table 6 (below) shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 units).
During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21 24°C, constant within ±1°C).

Reported statistics and error estimates:

Statistics are reported in appendix 2 and 3, appended to attached background material

TABLES

Table 1 - Growth Rate and Percentage Inhibition for the Total Test Period

Test solution	Mean	Std. Dev.	n	%Inhibition
Nominal conc. (µg/L)
Solvent control	1.93	0.0216	6
1	1.991	0.0331	3	-3.2
10	1.989	0.0198	3	-3.1
100	1.981	0.0376	6	-2.6

Table 2 - Growth Rate and Percentage Inhibition at Different Time Intervals

Test solution	n	0 – 24 h		24 – 48 h		48 – 72h
Nominal conc. (µg/L)		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Solvent control	6	2.606		1.536		1.646
100	6	2.664	-2.2	1.77	-15	1.507	8.5

Table 3 - Yield and Percentage Inhibition for the Total Test Period

Test solution	Mean	Std. Dev.	n	%Inhibition
Nominal conc. (µg/L)
Solvent control	326.235	21.044	6
1	393.217	38.371	3	-21
10	389.274	22.8269	3	-19
100	381.647	40.5267	6	-17

Table 4 - Effect Parameters Based on Mean Measured Concentrations

Parameter (µg/L)	NOEC	EC10	EC50
Growth rate	57	>57	>57
Yield	57	>57	>57

Table 5 - Effect Parameters Effect Parameters Based on Loading Rates

Parameter (µg/L)	NOEC	EC10	EC50
Growth rate	57	>57	>57
Yield	57	>57	>57

Table 6 - pH Levels Recorded during the Test

Test solution	pH
Nominal conc. (µg/L)	t=0h	t=72h
Blank control	7.9	8.1
Solvent control	8	8.1
100	8	8

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of the test item.

The 72h-EC50 for growth rate inhibition (ERC50) and yield inhibition (EYC50) exceeded the maximum solubility of the test item in test medium, i.e. exceeded a mean measured concentration of 57 µg/L.
The 72h-EL50 for growth rate inhibition (ERL50) and yield inhibition (EYL50) exceeded the maximum solubility of the test item in test medium, i.e. exceeded a loading rate of 100 µg/L.

The 72h-NOEC for both growth rate inhibition and yield inhibition was 57 µg/L.
The 72h-NOELR for both growth rate inhibition and yield inhibition was 100 µg/L.

Due to the very low solubility of the test item in water, concentration levels that might be toxic for algae could not be reached.

Executive summary:

The objective of the study was to evaluate the test item for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOEC, EC10, EC20 and EC50 for both inhibition of growth rate and inhibition of yield.

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 201 and OECD GD 23
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	6 replicates per control group and for the highest test concentration, 3 per intermediate test group.
Sampling for analysis	At t=0 and t=72hours.
Experimental conditions
pH and temperature	Within the ranges specified in OECD TG 201
Light regime	Continuous
Results
Actual exposure concentrations	57 µg/L (Mean measured concentration in limit concentration)
Recorded effects	At the end of the test, cell densities in both control groups were below the cell densities measured for the test item exposed groups, indicating a stimulation rather than an inhibition of algal growth due to test item exposure.
	Statistical comparison of algal growth between the blank and solvent control indicated that a significant difference was present for both growth rate and yield. Therefore, the solvent control was used as a reference to determine growth rate and yield inhibition at the different test item concentrations.
	No statistically significant inhibition was recorded for growth rate or yield at any of the concentrations tested when compared to the solvent control group.

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

Parameter (µg/L)	NOEC	EC10	EC50
Growth rate	57	>57	>57
Yield	57	>57	>57

Parameter (µg/L)	NOELR	EL10	EL50
Growth rate	100	>100	>100
Yield	100	>100	>100

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for freshwater algae:

57 µg/L

Additional information

The 72h-EC50 for growth rate inhibition (ERC50) and yield inhibition (EYC50) exceeded the maximum solubility of the test item in test medium, i.e. exceeded a mean measured concentration of 57 µg/L.

The 72h-EL50 for growth rate inhibition (ERL50) and yield inhibition (EYL50) exceeded the maximum solubility of the test item in test medium, i.e. exceeded a loading rate of 100 µg/L.