4-[bis(phenylsubstituted)substituted]phthalic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 03 November 2017; Experimental completion date: 06 May 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

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:

Preliminary Stability Analyses:
Analysis of the range-finding test preparations which had been stored frozen for a prolonged period prior to analysis showed measured test concentrations of less than the LOQ were obtained. It was therefore considered appropriate to perform some additional stability analyses with immediate analysis.
A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 500 mL to give a 100 mg/L test solution. A series of dilutions was made from this solution to give further test solutions of 10 and 1.0 mg/L. A sample of each test preparation was taken for immediate analysis. A further sample of each test preparation was incubated under test conditions for a period of 72 hours prior to analysis. All samples were stabilized with the addition of 100 µL of formic acid.

Total Organic Carbon Analysis:
Samples from the definitive test were taken from the uninoculated control and each test group at 0 and 72 hours for TOC analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.


Verification of Test Concentrations:
Chemical analysis of both the range-finding test and preliminary stability test samples showed variable results were obtained. It was considered likely that the salts in the culture medium were interfering with the analysis and as such compound specific analysis was considered inappropriate for this test item.

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. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/L for a period of 72 hours.

A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 20 minutes and the volume adjusted to 500 mL to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (4.0 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.

The stock solutions and each prepared concentration were 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 (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 30 minutes and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg/L. An aliquot (300 mL) of each of the stock solutions was separately inoculated with algal suspension (1.4 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.

The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

A sample of each test concentration containing no algal cells was incubated alongside the test to provide samples for TOC analysis at 0 and 72 hours.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained under constant agitation by orbital shaker (apprixiamtely 150 rpm) and constant illumination at 24 ±1 ºC.

Prior to the start of the test sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 ºC until the algal cell density was approximately 10^4 to 10^5 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

Temperature was maintained at 24 ±1 ºC throughout the test.

pH:

The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.8 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines.
A concentration dependent decline in pH was observed in the test preparations at the start of the test in the range of pH 7.5 at 1.0 mg/L to pH 3.3 at 100 mg/L. Given that this was considered to be an intrinsic property of the test item, no adjustments were made to the pH of the test preparations prior to inoculation with algae at the start of the test.

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

Total Organic Carbon (TOC) analysis of the 3.2, 10, 32 and 100 mg/L test preparations at 0 hours showed measured carbon concentrations to range from 1.8 to 48 mg/L, equivalent to dissolved test item concentrations of 3.7 to 97 mg/L. There was no significant change in the measured carbon concentrations obtained at 72 hours (1.7 to 48 mg/L; equivalent to dissolved test item concentrations of 3.5 to 96 mg test item/L). Analysis of the 1.0 mg/L test preparations at 0 and 72 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). However, as all other concentrations were within the acceptable limits it was considered appropriate to calculate the results based on nominal concentrations only.

Details on test conditions:

Range-Finding Test:
The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were used for each control and test concentration. The test item was dissolved directly in culture medium.
The control group was maintained under identical conditions but not exposed to the test item.
At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter® Multisizer Particle Counter. The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
After 72 hours the cell density of each flask was determined using a Coulter® Multisizer Particle Counter.

Definitive Test:
As in the range-finding test 250 mL glass conical flasks were used. Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Pre-culture conditions gave an algal suspension in log phase growth characterized by a cell density of 1.07 x 10^6 cells per mL. Inoculation of 300 mL of test medium with 1.4 mL of this algal suspension gave an initial nominal cell density of 5.00 x 10^3 cells per mL and had no significant dilution effect on the final test concentration.
The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ±1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Assessments:
Test Organism Observations:
Samples were taken at 23, 46 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10^3 cells/mL) was taken as the starting cell density.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Water Quality Criteria:
The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
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:

72 h

Dose descriptor:

EC50

Effect conc.:

20 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Range-finding Test:
The results showed no significant effect on growth rate at the test concentrations of 0.10, 1.0 and 10 mg/L. However, growth was observed to be reduced at 100 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Definitive Test:
The growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72 Hour exposure period.

Inhibition of Growth Rate:
ErC10 (0 to 72 hour): 9.7 mg/L
ErC20 (0 to 72 hour): 13 mg/L
ErC50 (0 to 72 hour): 20 mg/L; 95% confidence limits 17 to 24 mg/L
where ErCx is the test concentration that reduced growth rate by x%.
Statistical analysis of the growth rate data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955). There were no statistically significant differences (P0.05), between the control, 1.0, 3.2 and 10 mg/L test concentration, however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on growth rate was 10 mg/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour): 3.2 mg/L
EyC20 (0 to 72 hour): 4.9 mg/L
EyC50 (0 to 72 hour): 10 mg/L; 95% confidence limits 8.0 to 13 mg/L
Where EyCx is the test concentration that reduced yield by x%.

There were no statistically significant differences (P≥0.05), between the control and 1.0 mg/L test concentration however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on yield was 1.0 mg/L. Correspondingly the LOEC based on yield was 3.2 mg/L.

Results with reference substance (positive control):

A positive control (Envigo study number RD71YQ) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour): 1.6 mg/L; 95% confidence limits 1.4 to 1.8 mg/L
EyC50 (0 to 72 hour): 0.77 mg/L; 95% confidence limits 0.68 to 0.87 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Any other information on results incl. tables

Validation Criteria

The following data show that the cell concentration of the control cultures increased by a factor of 382 after 72 hours. This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.

Nominal cell density of control at 0 hours:          5.00 x 10³cells per mL

Mean cell density of control at 72 hours:          1.91 x 10⁶cells per mL

The mean coefficient of variation for section by section specific growth rate for the control cultures was 12% and hence satisfied the validation criterion given in the OECD Guideline which states the mean must not exceed 35%.

The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hour) was 2% and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7%.

 Observations on Cultures

   

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10 mg/L, however no cells were observed to be present in the test cultures at 32 and 100 mg/L.

Observations on Test Item Solubility

At the start of the test all control cultures were observed to be clear colorless solutions. The test cultures were observed to range from clear and colorless at 1.0 mg/L through to yellow at 100 mg/L. After the 72‑Hour test period all control, 1.0, 3.2 and 10 mg/L test cultures were green dispersions whilst the 32 mg/L test cultures were extremely pale yellow solutions and the 100 mg/L test cultures were yellow solutions.

Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition*
Control	R1	0.083	-	2.02E+06	-
	R2	0.083		2.00E+06
	R3	0.080		1.62E+06
	R4	0.084		2.06E+06
	R5	0.084		2.05E+06
	R6	0.081		1.67E+06
	Mean	0.083		1.90E+06
	SD	0.002		2.02E+05
1.0	R1	0.084	[1]	2.08E+06
	R2	0.083	0	1.97E+06
	R3	0.082	1	1.88E+06
	Mean	0.083	0	1.97E+06	[4]
	SD	0.001		1.01E+05
3.2	R1	0.080	4	1.56E+06
	R2	0.079	5	1.45E+06
	R3	0.079	5	1.48E+06
	Mean	0.079	5	1.50E+06	21
	SD	0.001		5.81E+04
10	R1	0.074	11	1.03E+06
	R2	0.074	11	1.06E+06
	R3	0.076	8	1.20E+06
	Mean	0.075	10	1.10E+06	42
	SD	0.001		9.09E+04
32	R1	0.031	63	4.08E+04
	R2	0.011	87	6.03E+03
	R3	0.008	90	3.95E+03
	Mean	0.017	80	1.69E+04	99
	SD	0.013		2.07E+04
100	R1	0.006	93	2.51E+03
	R2	0.005	94	2.27E+03
	R3	0.005	94	2.22E+03
	Mean	0.005	94	2.33E+03	100
	SD	0.001		1.55E+02

 

*In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R   = Replicate

SD   =Standard Deviation

[ ]    =Increase in growth compared to controls

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72 Hour period and gave the following results:

Growth Rate:
EC50: 20 mg/L (95% CL: 17 - 24 mg/L)
NOEC: 10 mg/L
LOEC: 32 mg/L

Yield:
EC50: 10 mg/L (95% CL: 8.0 - 13 mg/L)
NOEC: 1.0 mg/L
LOEC: 3.2 mg/L

Executive summary:

 Introduction

A study was perford to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) No 761/2009.

Methods

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter^®Multisizer Particle Counter.

Results.

Total Organic Carbon (TOC) analysis of the 3.2, 10, 32 and 100 mg/L test preparations at 0 hours showed measured carbon concentrations to range from 1.8 to 48 mg/L, equivalent to dissolved test item concentrations of 3.7 to 97 mg/L. There was no significant change in the measured carbon concentrations obtained at 72 hours (1.7 to 48 mg/L; equivalent to dissolved test item concentrations of 3.5 to 96 mg test item/L). Analysis of the 1.0 mg/L test preparations at 0 and 72 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). However, as all other concentrations were within the acceptable limits it was considered appropriate to calculate the results based on nominal concentrations only.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Response Variable	EC50 (mg/L)	95% Confidence Limits (mg/L)			No Observed Effect Concentration (NOEC) (mg/L)	Lowest Observed Effect Concentration (LOEC) (mg/L)
Growth Rate	20	17	-	24	10	32
Yield	10	8.0	-	13	1.0	3.2