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

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20.03.2015 - 30.04.2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

adopted 1984

Qualifier:

according to guideline

Guideline:

other: Mlinistry of Environmental protection of the peoples Republic of China, The Guidenes for the Testing of Chernicas Effects on Biotic systems, 2013, 207, Earth Worm, Acute Toxicky Test.

GLP compliance:

yes

Specific details on test material used for the study:

N/A

Analytical monitoring:

no

Details on sampling:

N/A

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

- Method of mixing into soil: The batch of the test item tested was a clear colourless liquid with a purity of > 99%. Since the test substance was insoluble in Milli-RO water (tap water purified by reverse osmosis), acetone (≥ 99.8%) was used as a solvent. Solubility in Milli-RO water and organic solvent was based on a pre-test performed at NOTOX B.V. Preparation started with a stock solution prepared at a loading rate of 4 g test substance per 10 mL acetone (stock A). No special treatment other than careful mixing was necessary to dissolve the test substance. Stocks B through E were subsequently diluted (0.5 mL of higher stock was made up to 5 mL with acetone) from stock A through D, respectively. The colour of the stocks ranged from clear and yellow in stock A, thought clear and slight yellow in stock B to clear and colourless in stocks C to E. Subsequently, 1.45 mL of stocks B to E were spiked on 10 g silver sand. For the highest concentration 5.24 mL of stock A was spiked on 40 g of sand and the solvent control received 5.24 mL of acetone on 40 g sand. Each sand - test substance mixture was thoroughly mixed and subsequently left for to allow the solvent to evaporate. Soil pH was set with 11.4 g CaCO3 per kg of artificial soil (Merck, Darmstadt, Germany). Spiked quartz sand was added to artificial soil (dry artificial soil, CaCO3and Milli-RO water mixed for 10 minutes) and the whole was mixed for another 10 min using a mixing device.
- Controls: solvent control with acetone
- Chemical name of vehicle: acetone
- Evaporation of vehicle before use: yes

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Earthworm (Eisenia fetida)
- Source: Shanghai Yinquan Agriculture Development Co., Ltd (ID: EF-MKT-141127)
- Age at test initiation: adult
- Weight at test initiation: 300 - 600 mg

BREEDING CONDITIONS
- Method of breeding: Earthworms are bred on a substrate consisting of 50% by volume peat and 50% by volume cow manure and kept under 20±2℃ for 7 days prior to testing. The test organisms were with clitellum and were transferred into artificial soil (without test substance) approximately 24 hours prior to the start of the test. At the start of the test, the adult earthworms used originated from the same population, were with clitellum and wet weight of 300 to 600 mg.

- Feeding: no food during exposure

ACCLIMATION
- Acclimation period: 14 d

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Remarks:

N/A

Post exposure observation period:

N/A

Test temperature:

21.3 - 22 °C

pH:

6.15 to 6.2 at test start
6.11 to 6.15 at test end

Moisture:

control: 24.36 - 24.57 %
solvent control: 23.36 - 24.00 %
tratements 21.32 - 23.24 %

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1.0 L glass with lid
- Amount of soil or substrate: 750 g/vessel
- No. of organisms per container (treatment, control and solvent conrol): 10
- No of replicates: 4
- climate chamber at 21.3 to 22.0 °C for a period of 14 days

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Artificial soil as described in the guidelines: 10% sphagnum peat, 20% kaolinite clay, 70% Silica sand (screened with 0.2 mm sieve); pH was adjusted to 6.0 +- 0.5 by addition of calcium carbonate.
- Water content: 25 - 42% of the dry weight

OTHER TEST CONDITIONS
- Photoperiod: continuous light
- Light intensity: 508-581 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Mortality and clinical effects were observed on day 7 and 14 of exposure.
- Ten earthworms were weighed individually prior to the start of the test. For all vessels the cumulative weight of the ten earthworms per vessel was recorded. The latter was also done for all vessels at the end of the test.

SOLVENT CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: control, solvent control, 10, 100 and 1000 mg/kg soil dw (nominal moisture 40-60 %) without replicates)
- Results used to determine the conditions for the definitive study: No mortality or other effects occurred up to the highest test concentration.

Nominal and measured concentrations:

Nominal: control, solvent control and 1000 mg/kg soil dw

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Key result

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: No mortalities occurred in the control group, solvent control group and treatment group at the 7th but 14th day mortality rate was 2.5% for all groups. Compared with the control groups, the solvent control group and 1000mg/kg group had no significant difference at the end of the test.
- Total mass of adults at beginning of test: For weight, compared with the control groups, 1000 mg/kg treatment group was significantly different (According to the result of Dunnett's Test, p=0.031, p<0.05) of the variance t-test at the 14th day. Compared with the control groups, the solvent control group had no significant difference (According to the result of Homoscedastic t-test, p =0.51, p>0.05) of the variance t-test at the end of the test. The treatment group lost less weight than the controls. The significant difference was because the treatment group lost less weight than the control group. There was no adverse effect.

- Behavioural and pathological symptoms: None

Results with reference substance (positive control):

- Results with reference substance valid? yes
The LC50 of the toxic reference substance chloroacetamide was determined occasionally following “Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals-Effects on Biotic Systems, 2013, 207 Earthworm, Acute Toxicity Test” and “OECD, Guidelines for the Testing of Chemicals, 1984, 207 Earthworm, Acute Toxicity Test”. The most recent test with chloroacetamide was performed from 8th July. 2014 to 22th July. 2014 (14 days test). In this study, the reference substance was tested at 5 concentrations of 3.75, 7.50, 15, 30 and 60 mg/kg dry artificial soil weight. The mortality of the chloroacetamide treatment group were shown to be between 2.5% to 100% (see Appendix 3). The 14-days LC50 for chloroacetamide was 13 mg/kg dry soil weight.
Test from this reference substance conducted over 5 years in the laboratory and show that the range of LC50 is between 10 to 31 mg/kg, so the 13 mg/kg was an acceptable value.

Reported statistics and error estimates:

The differences of the average body weight of the surviving earthworms between the control group and the treatment groups were determined by using the Homoscedastic t-Test and Dunnett's at the end of the study. The data analyses were carried out with Toxcalc software v5.0.32.

Table 3: Range-finding test: Mortality of Eisenia fetida exposed to the test substance and control at 7 and 14 days after exposure in the range-finding test

Parameter	Blank control		Solvent control		Treatment groups  (mg/kg dry soil weight)
					10		100		1000
Duration (d)	7	14	7	14	7	14	7	14	7	14
Number of earthworms	10	10	10	10	10	10	10	10	10	10
Number of mortality	0	0	0	0	1	1	0	0	0	0
Mortality (%)	0	0	0	0	2.5	2.5	0	0	0	0

Table 4: Main test: The mortality of Eisenia fetida exposed to the test substance and control at 7 and 14 days after application in limit test

Parameter	Control		Solvent control		Treatments (1000 mg/kg dry soil weight)
Duration (d)	7	14	7	14	7	14
Number of earthworm	40	40	40	40	40	40
Number of mortality (A replicate)	0	0	0	0	0	0
Number of mortality (B replicate)	0	1	0	1	0	1
Number of mortality (C replicate)	0	0	0	0	0	0
Number of mortality (D replicate)	0	0	0	0	0	0
Total	0	1	0	1	0	1
Mortality (%)	0	2.5	0	2.5	0	2.5

Validity criteria fulfilled:

yes

Remarks:

2.5% of the earthworms died in the control group, and 2.5% of the earthworms died in the solvent control group. Thus, the test fullfills the validity criterion that control mortality should not exceed 10%.

Conclusions:

The acute toxicity of the test substance 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters to Eisenia fetida was determined in a 14 days artificial soil test according to OECD 207.

Based on the results of range-finding test, one treatment group and one blank control group, one solvent control group which had four replicates of 10 worms per concentration were included in definitive test. The nominal concentrations of the treatment groups were 1000 mg/kg dry artificial soil weight. The number of mortalities were determined after 7 and 14 days exposure.

Mortality of the earthworms in control group was 2.5%, and in solvent control group was 2.5% which was not more than 10%, so the test was valid. Under conditions of the test, the 7-Day LC50 and the 14-Day LC50 for the test substance to earthworms (Eisenia fetida) based on nominal test concentrations was more than 1000 mg/kg dry soil weight.

Executive summary:

Expressed as nominal test substance concentration, the median lethal concentrations of 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters on Eisenia fetida are as follows:

The 7-day LC50 value was more than 1000 mg/kg dry soil weight.

The 14-day LC50 value was more than 1000 mg/kg dry soil weight.

No mortalities occurred in the control group, solvent control group and treatment group at the 7th but 14th day mortality rate was 2.5% for all groups.Compared with the control groups, the solvent control group and 1000 mg/kg group had no significant difference at the end of the test.

For weight, compared with the control groups, 1000 mg/kg treatment group was significantly different (According to the result of Dunnett's Test, p=0.031, p<0.05) of the variance t-test at the 14th day. Compared with the control groups, the solvent control group had no significant difference (According to the result of Homoscedastic t-test, p =0.51, p>0.05) of the variance t-test at the end of the test.

The measured pH values and soil moisture content in the control, solvent control and test cultures were given in 19.4. pH values were 6.15 to 6.20 at initiation and 6.11 to 6.15 at termination. Soil moisture content in the control, solvent control and test groups were 23.24 to 24.36 at initiation and 21.32 to 24.57 at termination.

The measured temperature of climate chamber remained at 21.6 to 22.0℃ throughout the study, which should be in the range of 18-22℃. The light intensity was 486-567 lux which should be in the range of 400-800 lux.

Description of key information

The acute toxicity of the test substance 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters to Eisenia fetida was determined in a 14 days artificial soil test according to OECD 207.

Based on the results of range-finding test, one treatment group and one blank control group, one solvent control group which had four replicates of 10 worms per concentration were included in definitive test. The nominal concentrations of the treatment groups were 1000 mg/kg dry artificial soil weight. The number of mortalities were determined after 7 and 14 days exposure.

Mortality of the earthworms in control group was 2.5%, and in solvent control group was 2.5% which was not more than 10%, so the test was valid. Under conditions of the test, the 7-Day LC50 and the 14-Day LC50 for the test substance to earthworms (Eisenia fetida) based on nominal test concentrations was more than 1000 mg/kg dry soil weight.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw