1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

19 June 2013 to 11 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted in accordance with OECD and EU Guidelines.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Main test: nominal loading rate of 100 mg/L. Range-finding test: nominal loading rates of 10 and 100 mg/L
- Sampling method: Main test: Samples were taken from the control and the 100 mg/L loading rate WAF test group (replicates R1 - R6, pooled) at 0 and 72 hours for quantitative analysis. Range-finding test: Samples of each loading rate WAF were taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration. Duplicate samples were taken at each occasion.
- Sample storage conditions before analysis: All samples were stored at approximately -20 °C prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).
- Eluate: None
- Differential loading: Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed.
- Controls: Yes - not exposed to test item. Positive control: potassium dichromate
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc): Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.

FURTHER DETAILS:
Range-finding test
Amounts of test item (20 and 200 mg) were each separately added to the surface of 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 47 hours and the mixtures allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.5 mL) to give the required test concentrations of IO and 100 mg/L loading rate WAF. The control group was maintained under identical conditions but not exposed to the test item.

Main test:
An amount of test item (250 mg) was added to the surface of 2.5 liters of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 47 hours and the mixture allowed to stand for 1 hour. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. An aliquot (1 liter) of the WAF was inoculated with algal suspension (7.1 mL) to give the required test concentration of 100 mg/L loading rate WAF. The control group was maintained under identical conditions but not exposed to the test item.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.
- Age of inoculum (at test initiation): Not specified.
- Method of cultivation: Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.

ACCLIMATION
- Acclimation period: Not specified. 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 E+3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 - 10 E+5 cells/mL.
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- Any deformed or abnormal cells observed: No data.

TEST SYSTEM:
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 E+3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 - 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10 E+4 - 10 E+5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not specified.

Test temperature:

Temperature of 24 ± 1 °C.

pH:

At 0 hours: pH = 7.7 to 7.9. At 72 hours: pH = 8.0.

Dissolved oxygen:

Not specified.

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal loading rate of 100 mg/L.
Measured: less than the limit of quantitation (LOQ): 0.00097 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass conical flasks
- Type: closed. Plugged with polyurethane foam bungs to reduce evaporation.
- Material, size, headspace, fill volume: Glass, 250 mL containing 100 mL of test preparation
- Aeration: Not specified.
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A, static
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: Initial nominal cell density of 5 x 10 E+3 cells per mL
- Control end cells density: 1.13 x 10 E+6 cells per mL. The cell concentration of the control cultures increased by a factor of 181 after 72 hours.
- No. of organisms per vessel: 5 x 10 E+5
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): N/A

GROWTH MEDIUM
- Standard medium used: No information.
- Detailed composition if non-standard medium was used:
NaNO3 25.5 mg/L
MgCl2.6H2O 12.16 mg/L
CaCl2.2H2O 4.41 mg/L
MgS04.7H2O 14.6 mg/L
K2HPO4 1.044 mg/L
NaHCO3 15.0 mg/L
H3BO3 0.186 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.00327 mg/L
FeCl3.6H2O 0.160 mg/L
CoCl2.6H2O 0.00143 mg/L
Na2MoO4.2H2O 0.00726 mg/L
CuCl2.2H2O 0.000012 mg/L
Na2EDTA.2H2O 0.30 mg/L
The culture medium was prepared using reverse osmosis purified deionized water* and the pH adjusted to 7.5 ± 0.1 with O.lN NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1 M NaOH or HCl.

OTHER TEST CONDITIONS
- Sterile test conditions: no information
- Adjustment of pH: no
- Photoperiod: under continuous illumination
- Light intensity and quality: Intensity approximately 7000 lux provided by warm white lighting (380 - 730 nm).
- Salinity (for marine algae): N/A
The pH of the control and the 100 mg/L loading rate WAF test preparation was determined at initiation of the test and after 72 hours exposure. The pH was measured using a WTW pH 320 pH meter. The temperature within the incubator was recorded daily.
Vortex Depth Measurements: The vortex depth was recorded at the start and end of the mixing period

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Coulter® Multisizer Particle Counter
- Chlorophyll measurement: No
- Other: Growth rates and yields were calculated from cell densities.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: One concentration in main test. Factor of 10 for dose range finding test.
- Justification for using less concentrations than requested by guideline: It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.
- Range finding study
- Test concentrations: 10 and 100 mg/L loading rate WAF.
- Results used to determine the conditions for the definitive study: The results showed no effect on growth at 10 and 100 mg/L loading rate WAF. Based on the result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
The cell concentration of the control cultures increased by a factor of 181 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. Mean cell density of control at 0 hours = 6.21 x 103 cells per mL. Mean cell density of control at 72 hours = 1.13 x 106 cells per mL.
- Observation of abnormalities (for algal test): All test and control cultures were inspected microscopically at 72 hours. There were no abnormalities detected in any of the control or test cultures.
- Any stimulation of growth found in any treatment: No. There were no statistically significant decreases in growth rate or yield (p=/>0.05), between the control and 100 mg/L loading rate WAF test group.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations on the test media were carried out during the mixing and testing of the WAF. At both the start and end of the mixing period, and following a 1-Hour standing period, the 100 mg/L loading rate WAF was observed to have formed a clear colorless media column with oily globules of test item on the media surface. Microscopic examination of the WAF showed there to be no micro-dispersions of test item present. At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures were observed to be green dispersions.
- Effect concentrations exceeding solubility of substance in test medium: Loading rate WAF reported due to low aqueous water solubility.

Physico-Chemical Measurements:
Temperature was maintained at 24 ± 1 °C throughout the test.
The pH value of the control cultures was observed to increase from pH 7.7 at 0 hours to pH 8.0 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.

Vortex Depth Measurements:
The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
ErC50 (0 - 72 h) = 1.2 mg/L; 95% confidence limits 1.0 - 1.3 mg/L
EyC50 (0 -72 h) = 0.52 mg/L; 95% confidence limits 0.45 - 0.62 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L
No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L
Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

Reported statistics and error estimates:

There were no statistically significant decreases in yield or growth (p=/>0.05), between the control and 100 mg/L loading rate WAF and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/L loading rate WAF.

Cell Densities and Percentage Inhibition of Growth from the Range-finding Test:

 

Nominal Loading Rate (mg/L)		Cell Densities (cells per mL)		Inhibition Values (%)
		0 Hours	72 Hours	Growth Rate	Yield
Control	R1	5.71E+03	6.05E+05	-	-
	R2	4.56E+03	6.81E+05
	Mean	5.14E+03	6.43E+05
10	R1	5.23E+03	8.22E+05	[6]	[29]
	R2	5.02E+03	8.33E+05
	Mean	5.13E+03	8.27E+05
100	R1	5.09E+03	9.17E+05	[9]	[59]
	R2	5.66E+03	l.12E+06
	Mean	5.38E+03	l.02E+06

*    Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 and R2= Replicates 1 and 2

[Increase in growth compared to controls]

Cell Densities and pH Values in the Definitive Test

 

Nominal Loading Rate (mg/L)		pH	Cell Densities (cells per mL)				pH
		Oh	Oh	24h	48h	72h	72h
Control	R1	7.7	6.30E+03	3.12E+04	l.94E+05	l.19E+06	8.0
	R2		5.79E+03	2.99E+04	l.71E+05	l.16E+06
	R3		5.82E+03	3.00E+04	l.50E+05	9.63E+05
	R4		6.06E+03	3.49E+04	l.96E+05	l.27E+06
	R5		6.16E+03	3.33E+04	l.88E+05	l.11E+06
	R6		7.09E+03	3.08E+04	l.77E+05	l.06E+06
	Mean		6.21E+03	3.17E+04	l.79E+05	l.13E+06
100	R1	7.9	6.22E+03	3.17E+04	2.01E+05	l.19E+06	8.0
	R2		5.79E+03	2.82E+04	2.06E+05	1.20E+06
	R3		4.98E+03	3.34E+04	2.10E+05	l.34E+06
	R4		4.98E+03	3.21E+04	l.86E+05	l.07E+06
	R5		5.10E+03	3.28E+04	2.00E+05	l.03E+06
	R6		4.83E+03	3.33E+04	l.79E+05	l.16E+06
	Mean		5.32E+03	3.19E+04	l.97E+05	l.17E+06

*Cell densities represent the mean number of cells per ml calculated from the mean of the cell counts from 3 counts for each of the replicate flasks.

R1 - 6 = Replicates 1 to 6

Daily Specific Growth Rates for the Control Cultures in the DefinitiveTest

 

		Daily Specific Growth Rate (cells/mL/hour)
		Day 0 -1	Day 1-2	Day 2 -3
Control	R1	0.076	0.076	0.076
	R2	0.074	0.073	0.080
	R3	0.075	0.067	0.077
	R4	0.081	0.072	0.078
	R5	0.079	0.072	0.074
	R6	0.076	0.073	0.075
	Mean	0.077	0.072	0.077

R1 - 6 = Replicates 1 to 6

Inhibition of Growth Rate and Yield in the Definitive Test

 

Nominal Loading Rate (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 -72h	%Inhibition	0 -72h	%Inhibition*
Control	R1	0.076		l.18E+06
	R2	0.076		l.16E+06
	R3	0.073		9.57E+05
	R4	0.077	-	l.26E+06	-
	R5	0.075		l.10E+06
	R6	0.074		l.05E+06
	Mean	0.075		l.12E+06
	SD	0.001		1.06E+05
100	R1	0.076	[1]	l.18E+06
	R2	0.076	[1]	l.19E+06
	R3	0.078	[4]	1.34E+06
	R4	0.075	0	l.07E+06
	R5	0.074	1	l.02E+06
	R6	0.076	[1]	l.16E+06
	Mean	0.076	[1]	l.16E+06	[4]
	SD	0.001		l.11E+05

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

R1 - 6 = Replicates 1 to 6

SD = Standard Deviation

Vortex Depth Measurements at the Start and End of the Mixing Period

 

	Nominal Loading Rate (mg/L)
	Control		100
	*	+	*	+
Height of Media Column (cm)	15	15	15	15
Depth of Vortex (cm)	-0.2	-0.2	-0.2	-0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present

* = Start of mixing period

+= End of mixingperiod

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Executive summary:

Introduction

A study was performed 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) 761/2009.

 

Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF).

 

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) 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

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

 

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantitation (LOQ) of the analytical method employed, which was determined to be 0.00097 mg/L, were obtained. This does not infer that no test item was in solution, just that any which was dissolved, was at a concentration of less than 0.00097 mg/L.

 

Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, and the dissolved test item was below the quantifiable limit of the analytical method, the results were based on nominal loading rates only.

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-02-01 to 1994-02-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

As the test substance was poorly soluble in water, a 1 g/L mixture with deionised water was prepared. This mixture was stirred for 18 hours and filtrated afterwards. The filtrate was analysed and showed an initial content of 3.9 mg/L DOC and was used as stock solution.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Scenedesmus subspicatus (CHODAT (86.81 SAG))
- Source (laboratory, culture collection): Institut für Wasser-, Boden- und Lufthygiene, Berlin; subsequently in-house breeding
- Method of cultivation: in sterile-aerated Erlenmeyer flasks on light benches


ACCLIMATION
- Acclimation period: A preculture is withdrawn from a stock culture by transfer 3 days prior to the beginning of the test from which preculture the test cultures are inoculated at a cell density of approximately 20 000 cells/mL.
- Culturing media and conditions (same as test or not): not mentioned
- Any deformed or abnormal cells observed: not mentioned

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

Growth/test medium chemistry as described in guideline 92/69/EEC (C.3.), no further details mentioned

Test temperature:

24 ± 2 °C

pH:

8.0 - 8.9 (start of test)
8.7 - 9.3 (end of test)

Dissolved oxygen:

not mentioned

Salinity:

according to guideline

Nominal and measured concentrations:

Nominal: 0.24 / 0.39 / 0.67 / 1.2 / 2.0 / 3.5 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile-aerated Erlenmeyes flasks
- Initial cells density: 2x10E4 cells/mL
- Control end cells density: 88x10E4 cells/mL
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8
- No. of vessels per vehicle control (replicates): not applicable


GROWTH MEDIUM
- Standard medium used: yes


TEST MEDIUM / WATER PARAMETERS
no details mentioned


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not adjusted
- Photoperiod: not specified
- Light intensity and quality: approx. 8000 lux, white


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cell numbers for exposure times of 24, 48, 72 h
- Determination of cell concentrations: The cell numbers were determined photometrically at a wavelenght of 685 nm
- Chlorophyll measurement: no
- Other: none


TEST CONCENTRATIONS
- Spacing factor for test concentrations: not mentioned
- Range finding study: not mentioned

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 3.5 mg/L

Nominal / measured:

nominal

Conc. based on:

dissolved

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no observations made
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

no reference substance tested

Reported statistics and error estimates:

no statistical evaluation performed

Table #1: Cell density data:

concentration [mg/L]	cell number (x 10E4 cells/mL) after
	0 h	24 h	48 h	72 h
control	2	13	28	88
0.24	2	13	34	100
0.39	2	13	33	98
0.67	2	13	32	97
1.2	2	13	35	109
2.0	2	13	34	104
3.5	2	14	38	117

                                                                                 

Table #2: Growth curves:

concentration [mg/L]	biomass (area under the growth curve)	inhibition of biomass [%]	growth rate µ (0 - 72 h)	inhibition of growth rate [%]
control	80	-	1.261	-
0.24	92	-15	1.304	-3.4
0.39	90	-12.5	1.297	-2.9
0.67	88.5	-10.6	1.294	-2.6
1.2	97.5	-21.9	1.333	-5.7
2.0	94	-17.5	1.317	-4.4
3.5	105.5	-31.9	1.356	-7.5

negative values indicating an activation of growth

Validity criteria fulfilled:

yes

Conclusions:

Not toxic under test conditions in the range of water solubility.

Executive summary:

The inhibitory effect of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters on the growth of the green alga Scenedesmus subspicatus, strain CHODAT (86.81 SAG) had been tested in accordance with OECD Guideline for Testing of Chemicals No.201 and EEC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC.

The test algae were exposed to 6 concentrations of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters, ranging from 0.24 to 3.5 mg/L. As the test substance was poorly soluble in water, a 1 g/L mixture with deionised water was prepared. This mixture was stirred for 18 hours and filtrated afterwards. The filtrate was analysed and showed an initial content of 3.9 mg/L DOC and was used as stock solution. The cultures were incubated on sterile ventilated Erlenmeyer flasks on light tables at 24±2 °C. Growth was monitored by determining the cell number photometrically at a wave length of 685 nm after 24, 48 and 72 hours. No inhibitory effects on the growth of the green alga were observed.

On the basis of cell growth, a mean effective concentration was calculated to be EbC50 (72 h) > 3.5 mg/L.

On the basis of the growth rate, a mean effective concentration was calculated to be ErC50 (0 -72 h) > 3.5 mg/L.

The NOEC value was >= 3.5 mg/L (on basis of cell growth).

Description of key information

In both available studies the test substance was not toxic under test conditions in the range of water solubility.

Additional information

In the key study the inhibitory effect of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters on the growth of the green alga Scenedesmus subspicatus, strain CHODAT (86.81 SAG) had been tested in accordance with OECD Guideline for Testing of Chemicals No.201 and EEC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC.

The test algae were exposed to 6 concentrations of 1,2,4-Benzenetricarboxylic acid, mixed decyl and octyl triesters, ranging from 0.24 to 3.5 mg/L. As the test substance was poorly soluble in water, a 1 g/L mixture with deionised water was prepared. This mixture was stirred for 18 hours and filtrated afterwards. The filtrate was analysed and showed an initial content of 3.9 mg/L DOC and was used as stock solution. The cultures were incubated on sterile ventilated Erlenmeyer flasks on light tables at 24±2 °C. Growth was monitored by determining the cell number photometrically at a wave length of 685 nm after 24, 48 and 72 hours. No inhibitory effects on the growth of the green alga were observed.

On the basis of cell growth, a mean effective concentration was calculated to be EbC50 (72 h) > 3.5 mg/L.

On the basis of the growth rate, a mean effective concentration was calculated to be ErC50 (0 -72 h) > 3.5 mg/L.

The NOEC value was 3.5 mg/L (on basis of cell growth at the highest concentration achievable).

 

In a supporting study the effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values of > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF (highest loading rate tested).

 

Therefore in both tests the test substance was not toxic under test conditions in the range of water solubility.