4-[bis(phenylsubstituted)substituted]phthalic acid

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:

Experimental start date: 19 March 2018; Experimental completion date: 17 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Remarks:

None that were considered to have affected to outscome of the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The concentration and stability of the test item in the test preparations were verified by Total Organic Carbon (TOC) analysis at 0 and 48 hours.

Test solutions

Vehicle:

no

Details on test solutions:

Range-finding Test:
The test concentrations to be used in the definitive test were determined by a preliminary range finding test.
In the range finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L.

A nominal amount of test item (50 mg) was dissolved in test water with the aid of ultrasonication for 20 minutes and the volume adjusted to 500 mL to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 0.10, 1.0 and 10 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Definitive Test:
Based on the results of the range finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.

A nominal amount of test item (200 mg) was dissolved in test water with the aid of ultrasonication for 20 minutes and the volume adjusted to 2 liters to give the 100 mg/L test concentration from which a series of dilutions was made to give further test concentrations of 1.0, 3.2, 10 and 32 mg/L.
Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using first instar Daphnia magna derived from in house laboratory cultures.
Adult daphnids were maintained in 150 mL glass beakers containing 100 mL Elendt M7 medium in a temperature controlled room maintaining the water temperature at 18 to 22 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Test temperature:

20 ºC to 21 ºC

pH:

concentration dependent differences in pH were observed throughout the test.

Dissolved oxygen:

there were no treatment related differences for oxygen concentration

Nominal and measured concentrations:

Range-finding Test
Nominal: 0.10, 1.0, 10 and 100 mg/L

Definitive Test
Nominal : 1.0, 3.2, 10, 32 and 100 mg/L
Geometric Mean Measured: 1.0, 2.5, 8.4, 20 and 76 mg/L

TOC analysis of the 3.2, 10, 32 and 100 mg/L test preparations at 0 hours showed measured carbon concentrations to range from 2.4 to 49 mg/L, equivalent to dissolved test item concentrations of 4.7 to 98 mg/L. TOC analysis of the aged or expired preparations at 48 hours showed measured carbon concentrations to range from 0.65 to 29 mg/L, equivalent to dissolved test item concentrations of 1.3 to 54 mg/L. Analysis of the 1.0 mg/L test preparations at 0 and 48 showed measured concentrations of less than the limit of quantification (LOQ) were obtained, which was considered to be 1.0 mg C/L (2.0 mg test item/L). Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data.

Details on test conditions:

Test Water:
Reconstituted water (Elendt M7 medium) used for both the range finding and definitive tests.

Range-finding Test:
In the range finding test five daphnids were placed in each test and control vessel and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Two replicate test and control vessels were prepared. Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded.
The control group was maintained under identical conditions but not exposed to the test item.
Samples were taken at 0 and 48 hours for TOC analysis. All samples were fortified with 100 µl of formic acid and stored frozen prior to analysis.

Definitive Test:
As in the range finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test five daphnids were placed in each test and control vessel at random, in the test preparations. Four replicate test and control vessels were prepared. The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room maintaining the water temperature at 18 to 22 °C with a maximum deviation of ±1 °C with a photoperiod of 16 hours light (between 200 and 1200 Lux) and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.

The control group was maintained under identical conditions but not exposed to the test item.

The test preparations were not renewed during the exposure period.

EFFECT PARAMETERS MEASURED:
Test Organism Observations:
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim within 15 seconds after gentle agitation.

Water Quality Criteria:
Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded at the start and termination of the test. The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
The light intensity during the light period was measured using an ATP Instrumentation Lux meter.
The appearance of the test media was recorded daily.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

30 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

71 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

20 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range finding Test:
No immobilization was observed at the test concentrations of 0.1 and 1.0 mg/L, however, immobilization was observed at 10 and 100 mg/L and in the control.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Total Organic Carbon (TOC) analysis of the test preparations in the range finding test showed results to be anomalous. The anomalous results obtained were considered to be caused by an instrument issue rather than incorrectly prepared test solutions as the 0-Hour results greatly exceeded nominal concentrations. As TOC analysis does not indicate stability it was considered acceptable to carry out the definitive test based on the biological results only.

Definitive Test
Analysis of the immobilization data by the Trimmed Spearman-Karber method at 24 hours and Probit analysis using linear maximum likelihood regression at 48 hours based on the geometric mean measured test concentrations gave the following results:
Time EC50 95% Confidence limits
(Hour) (mg/L) (mg/L)
24 71 54 - 94
48 30 Not determined due to the nature of the data

The No Observed Effect Concentration after 24 and 48 hours exposure was 20 mg/L. The Lowest Observed Effect Concentration after 24 and 48 hours exposure was 76 mg/L.
The slope and standard error of the response curve at 48 hours were 3.7(standard error = 0.063). Due to the nature of the data it was not possible to calculate the slope and error of response curve at 24 hours.

Sub-Lethal Effects
Sub lethal effects of exposure were observed in the control and test concentrations of 3.2, 10, 32 and 100 mg/L. These responses were reduced mobility, pale and covered in debris.

Validation Criteria
The test was considered to be valid given that no more than 10% of the control daphnids showed immobilization or other signs of disease or stress and that the oxygen concentration at the end of the test was equal to or greater than 3 mg/L in the control and test vessels.

Water Quality:
Throughout the test the light intensity was observed to be in the range 881 to 889 Lux.

Observations on Test Item Solubility:
At the start and throughout the test all control and 1.0 and 3.2 mg/L test solutions were observed to be clear colorless solutions. At the start and throughout the test the10 and 32 mg/L test solutions were observed to be clear slightly yellow solutions whilst the 100 mg/L test solutions were observed to be clear yellow solutions. 

Results with reference substance (positive control):

A positive control used potassium dichromate as the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L.

Exposure conditions for the positive control were similar to those in the definitive test.
Analysis of the immobilization data was carried out using the Binomial Distribution method at 24 hours and the Trimmed Spearman-Karber method at 48 hours. All statistical analysis was carried out using the ToxRat Professional computer software package with results based on the nominal test concentrations and gave the following results:

24 hour:
EC50: 0.79 mg/L (95% CL: 0.73 - 0.86 mg/L)
NOEC: 0.56 mg/L
LOEC: 1.0 mg/L

48 hours:
EC50: 0.75 mg/L (95% CL: 0.56 - 2.0 mg/L)
NOEC: 0.56 mg/L
LOEC: 1.0 mg/L

The NOEC is based upon equal to or less than 10% immobilization at this concentration.
The results from the positive control with potassium dichromate were within the normal range for this reference item*.

*The results of the inter laboratory tests and a Technical Corrigendum to ISO 6341 give a 24 Hour EC50 for potassium dichromate within the range 0.6 mg/L to 2.1 mg/L

Any other information on results incl. tables

The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration (mg/L)	Geometric Mean Measured Test Concentration (mg/L)
1.0	1.0
3.2	2.5
10	8.4
32	20
100	76

Cumulative Immobilization Data and Observations in the Range‑finding Test

Nominal Concentration (mg/L)	Observations (Initial Population: 5 Per Replicate)
	24 Hours				48 Hours
	Cumulative Immobilized Daphnia		Observations		Cumulative Immobilized Daphnia		Observations
	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2	Rep1	Rep2
Control	0	0	5N	5 N	1	1	4 N	4 N
0.10	0	0	5 N	5 N	0	0	5 N	5 N
1.0	0	0	5 N	5 N	0	0	5 N	5 N
10	0	0	5 N	5 N	0	1	5 N	4 N
100	2	4	3 N	1 N	3	5	2 N	AI

R      =Replicate

N      = Normal

AI     = All daphnia immobilized

Cumulative Immobilization Data and Observations in the Definitive Test

Nominal Concentration (mg/L)	24 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	0	0	0	0	0	0	5N	5 N	5 N	5 N
1.0	0	0	0	0	0	0	5 N	5 N	5 N	5 N
3.2	0	0	0	0	0	0	5 N	5 N	5 N	5 N
10	0	0	0	0	0	0	5 N	5 N	5 N	5 N
32	0	0	0	0	0	0	5 N	5 N	4 N 1 R	5 N
100	3	0	3	4	10	50	1 N 1 R	3 N 1 R 1 P	2 R	1 R
Nominal Concentration (mg/L)	48 Hours
	Cumulative Immobilized Daphnia (Initial Population: 5 Per Replicate)						Observations
	Rep1	Rep2	Rep3	Rep4	Total	%	Rep1	Rep2	Rep3	Rep4
Control	1	0	0	0	1	5	3 N 1 R	5 N	5 N	5 N
1.0	0	0	0	1	1	5	5 N	5 N	5 N	4 N
3.2	0	0	0	1	1	5	5 N	4 N 1 C	5 N	4 N
10	0	0	3	0	3	15	5 N	4 N 1 R	2 N	2 N 3 P
32	0	1	1	0	2	10	3 N 2 R	3 N 1 R	3 N 1 R	3 N 2 P
100	5	5	5	5	20	100	AI	AI	AI	AI

Rep    =    Replicate

N          =    Normal

R     =   Reduced mobility

P      =   Pale

C     =   Covered in debris

AI     =   All daphnia immobilized

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of freshwater invertebrate Daphnia magna to the test item has been investigated and gave the following results:

48 hour timepoint:
EC50: 30 mg/L
NOEC: 20 mg/L
LOEC: 76 mg/L

Executive summary:

 Introduction

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

 Methods

Following a preliminary range‑finding test, 20 daphnids (four replicates of five animals) were exposed to an aqueous solution of the test item at nominal concentrations of 1.0,3.2,10,32 and 100 mg/L for 48 hours at a temperature of 20 °C to 21 °C under static test conditions. The number of immobilized Daphnia were recorded after 24 and 48 hours.

Results

Samples of the control and 1.0, 3.2, 10, 32 and 100 mg/L test concentrations were taken at 0 (fresh media) and 48 hours (old media) for Total Organic Carbon (TOC) analysis. From the results obtainedand the known carbon content of the test item, the concentration of test item was calculated.

Analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from less than the limit of quantification (LOQ) to 98 mg/L. Analysis of the old or expired test item at 48 hours showed measured test concentrations to range from less than the LOQ to 58 mg/L.

Given this decline in measured test concentrations it was considered justifiable to base the results on the geometric mean measured test concentrations of the test media in order to give a "worst case" analysis of the data.

Exposure of Daphnia magna to the test item gave the following results:

Time Point (Hours)	EC50 (mg/L)	95% Confidence Limits (mg/L)	NOEC (mg/L)	LOEC (mg/L)
48	30	Not determined	20	76

NOEC=No ObservedEffect Concentration

LOEC =Lowest Observed Effect Concentration