6-tert-butyl-2,4-xylenol

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Data waiving:

other justification

Justification for data waiving:

other:

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

26 June - 29 August 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Study, conducted to current guideline and with all associated information included. Read across to structural analogue.

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

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):
Purity: 98.5%
Stability under storage conditions: Stable
Specific Gravity / Density: 0.95 - 0.99 g/cm3
Molecular weight: 376 - 404
PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 0.0015 Pa at 20 °C
- Water solubility (under test conditions): <0.0005 g/L at 20°C
- log Pow: 9.2
- pKa: Not determined as the techniques specified in OECD Guideline 112 are not applicable to water-insoluble substances.
- pH dependance on stability: No
OTHER PROPERTIES (if relevant for this endpoint)
- Fate and behaviour in the environment (where relevant to application patterns):

Air (%) Water (%) Soil (%) Sediment (%)
C7 derivatives 0.0741 - 0.12 5.84 - 7.73 58.2 - 63.5 30.4- 34.9
C8 derivatives 0.0912 - 0.149 6.65 - 9.32 61.5 - 63.1 28.2 - 31.8
C9 derivatives 0.104 - 0.182 8.03 - 12.5 68 - 72.8 14.5 - 24.1

Analytical monitoring:

not required

Details on sampling:

Not required.

Vehicle:

yes

Details on preparation and application of test substrate:

The batch of C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl)propionate tested was a clear yellow viscous liquid with a purity of 98.5%. Since the test substance was insoluble in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA), acetone (≥99.8% pure, Merck, Darmstadt, Germany) 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 to allow acetone to evaporate.

Then, the test media were prepared according to Table 1 (See below)

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, CaCO3 and Milli-RO water mixed for 10 minutes) and the whole was mixed for another 10 minutes using a mixing device. A small quantity of controls and 1000 mg/kg d.s. treatment were kept apart for soil moisture and pH determination. The appearance of all test media was normal (similar to the control).

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Species: Earthworm (Eisenia fetida fetida).

Source: Earthworms were bred at the NOTOX B.V. facilities.

Age and weight of earthworms: four months old with clitellum, with an individual wet weight of 420 ±
90 mg (replicate C of the acetone control).

Reason for selection: This system has been internationally accepted.

Validity of batch: Mortality within 24 hours prior to the test was less than 10%.

Breeding
Start of each batch: With adult earthworms with well developed clitella, by placing 100 of them into ca. 15 litres of culture medium in a plastic container. Adults were removed between day 14 and 28 and juveniles were allowed to hatch from the cocoons produced by the adults.

Batches were split up and fresh culture medium was added in case of overcrowding. Worms were transferred to fresh medium regularly.

Composition of culture medium (based on dry mass):
Mixture of 40% sphagnum peat (Lentse Potgrond, Lent) and 60% pot soil (source Praxis, ‘s-Hertogenbosch) humidified with tap water. The pH of the mixture is 6.0 ± 1.0. When necessary, the medium was humidified with tap water during breeding. All sources are in The Netherlands.

Temperature: 20 ± 4°C.

Feeding: Every 7-10 days with oat meal (Quaker, PepsiCo International, Utrecht, The Netherlands).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Post exposure observation period:

None

Test temperature:

20 ± 2°C

pH:

6.0 ± 0.5 at the start of the test

Moisture:

25 – 42% of the dry weight

Details on test conditions:

Test duration: 14 days.

Test vessels: Approximately 1.8 litre, glass, covered with a glass lid.

Medium: Artificial soil as described in the guidelines:
-10% sphagnum peat (Lentse Potgrond, Lent, The Netherlands);
- 20% kaolin clay (Silex Pottenbakkermat., 's-Hertogenbosch, The Netherlands);
- 70% industrial sand M32 (Eurogrit, Nieuwegein, The Netherlands);
pH was adjusted by addition of calcium carbonate.

Number of earthworms: 10 earthworms each at concentrations of 0.1, 1.0, 10 and 100 mg/kg d.s. and 40 earthworms for the control and limit concentration (1000 mg/kg d.s.).

Loading:10 earthworms per vessel (containing approximately 500 grams of dry mass of the artificial soil).

Light: Continuous light.

Temperature: 20 ± 2°C

pH : 6.0 ± 0.5 at the start of the test

Aeration: No aeration

Water content of the medium: 25 – 42% of the dry weight

Acclimatization: Approximately 24 hours prior to the test, earthworms were acclimatized to the artificial soil.

Feeding: No feeding from the start of acclimatization until the end of the test period.

Introduction of earthworms After preparation of the test media the earthworms were washed with tap water and introduced onto the surface of the medium.

Measurements and recordings
Mortality and other effects After 7 and 14 days following the start of exposure, earthworms were checked for any obvious behavioural or pathological symptoms. Dead animals may decompose during the test, and for this reason any 'missing' animals were presumed and counted as dead.

Earthworm weight: 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.

pH and water content of the soil: At the start (prior to exposure) and at the end of the test (day 14) for the control and the highest test concentration.

Temperature of incubator: Continuously in a temperature control vessel.

Illumination: Measured on day 0 and 14.

Nominal and measured concentrations:

0.1, 1.0, 10, 100 and 1000 mg/kg dry soil.

Reference substance (positive control):

yes

Remarks:

2-Chloracetamide

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:

No mortality or any visual effects (e.g. avoidance behaviour, stiffness, flaccidity, coiling) in the earthworms were recorded at any concentration throughout the whole 14-day period of exposure.
Tabulated data is detailed below:

Results with reference substance (positive control):

The results of a reference test with 2-Chloracetamide (Merck, analytical grade) are appended to the report. Due to the stability of the sensitivity of the earthworms bred at NOTOX B.V., this reference test is performed once a year. The results of the most recent test are as follows:

REFERENCE TEST
Start: 05 January 2012
End: 19 January 2012

A 14-day acute toxicity study in the earthworm Eisenia fetida fetida with 2-Chloroacetamide (NOTOX project 498544).

The study procedures described in this report were based on the OECD guideline for Testing of Chemicals No. 207 (adopted April 4, 1984).
In addition, the procedure was designed to generally meet the test methods prescribed by the Council regulation (EC) No 440/2008 of 30 May 2008, EEC publication no. L 142, C.8 and the International Standard ISO 11268-1, Part 1, 1993.

The batch of 2-Chloroacetamide tested was a white crystalline powder with a purity of 99.9% (GC, area%) and was sufficiently soluble in Milli-RO water to allow addition in the form of a stock solution.

Earthworms of the species Eisenia fetida fetida were exposed to 10, 18, 32, 56 and
100 mg 2-Chloroacetamide per kg dry soil (further called d.s.). Milli-RO water was used as a vehicle. For each concentration and the control 40 earthworms were used (10 per replicate, 4 replicates). The control consisted of OECD-medium without test substance. Test duration was 14 days.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

2-Chloroacetamide caused no mortality at or below 56 mg/kg d.s. (NOEC).

The 14-day LC50 was 75 mg/kg d.s. with a 95% confidence interval ranging from 56 to
100 mg/kg d.s.

No statistically significant reduction of the body weight was recorded at or below 56 mg/kg d.s. (NOEC).

With the LC50 of 75 mg/kg d.s. the sensitivity of the earthworms used in this study matches the requirement of the ISO standard 11268-1 (i.e. 20-80 mg/kg d.s.).

The raw data and report from this study are kept in the NOTOX archives. The test described above was performed under GLP-conditions with a QA-check.

Reported statistics and error estimates:

None

Mortality

The mortality data are presented in Table2. No mortality or any visual effects (e.g. avoidance behaviour, stiffness, flaccidity, coiling) in the earthworms were recorded at any concentration throughout the whole 14-day period of exposure.

 

Table 2           Mortality and clinical effects

Concentration Test substance^1 (mg /kg d.s.)	Vessel	Initial number of earthworms	Cumulative mortality		Total percent mortality on day 14	Group mean percent mortality
			day 7	day 14
Solvent-control	A	10	0	0	0	0
	B	10	0	0	0
	C	10	0	0	0
	D	10	0	0	0
0.1	A	10	0	0	0
1.0	A	10	0	0	0
10	A	10	0	0	0
100	A	10	0	0	0
1000	A	10	0	0	0	0
	B	10	0	0	0
	C	10	0	0	0
	D	10	0	0	0

1. C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl)propionate

 

Bodyweight

Table 3 presents mean weights per replicate at the start and at the end of exposure. Individual weight of 10 earthworms was determined at the start of the test was 420± 90 mg, which was within the protocolled range (300-600 mg).

 

In the control the reduction in mean earthwormweightwas below 20% (2.8%).

 

A statistically (p<0.05) significant reduction in mean earthworm weight (12%) was found at the highest concentration tested, i.e. 1000 mg/kg d.s. However, this reduction was considered not to be biologically significant (<20%). Therefore, the NOEC for body weight reduction was set to 1000 mg/kg d.s.

 

Table 3           Earthworm body weight

Concentration Test substance^1 (mg /kg d.s.)	Vessel	Day 0			Day 14			Weight reduction  (%)	Group weight reduction  (%)
		Number of worms	Total weight (g)	Mean mass (mg)	Number of worms	Total weight (g)	Mean mass (mg)
Solvent-control	A	10	3.77	377	10	3.76	376	0.3	2.8
	B	10	4.06	406	10	3.97	397	2.2
	C	10	4.22	422	10	3.97	397	5.9
	D	10	4.36	436	10	4.23	423	3.0
0.1	A	10	3.79	379	10	3.63	363	4.2
1.0	A	10	3.96	396	10	3.94	394	0.5
10	A	10	4.20	420	10	4.02	402	4.3
100	A	10	4.36	436	10	4.08	408	6.4
1000	A	10	3.80	380	10	3.39	339	10.8	12*
	B	10	4.01	401	10	3.49	349	13.0
	C	10	4.07	407	10	3.70	370	9.1
	D	10	4.53	453	10	3.87	387	14.6

1. C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl)propionate

*=significantly different from the control (a=0.05).

 

Effect Parameter

Table 4 shows the effect parameters based on nominal concentrations.

 

Table 4            Effect parameters

Parameter	Concentration C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl)propionate (mg/kg d.s.)
14-day LC50	> 1000
NOEC for mortality	1000
NOEC for body weight reduction	1000

 

Experimental Conditions:

The temperature of the medium was between 19.4 and 21.0°C during the test. All experimental conditions were within the protocol-allowed ranges throughout the study.Table5summarizes the environmental conditions in the combined limit/range-finding test.

 

Table5            Experimental conditions

Parameter	Day 0	Day 14
Light intensity (lux)	515-654	648 - 611
Soil moisture (%)
Solvent - control	35	35
1000 mg/kg d.s.	34	35
pH
Solvent - control	6.5	7.4
1000 mg/kg d.s.	6.5	7.4

 

Validity criteria fulfilled:

yes

Conclusions:

Read across to the structural analogue C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl)propionate. Under the conditions of the present study C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl)propionate did not caused mortality of Eisenia fetida fetida at 1000 mg/kg d.s after 14 days of exposure (NOEC).

The 14-day LC50 was beyond the range tested, i.e. exceeded the limit concentration of 1000 mg/kg d.s.

The 14-day EC50 for weight reduction was beyond the range tested, i.e. exceeded the limit concentration of 1000 mg/kg d.s.

Executive summary:

Read across to the structural analogue C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl)propionate. Given that it is recommended as RMM that CAS1879-09-0 is not released to the environment, this use of read across is considered to be an appropriate way to address the terrestrial endpoint. As such, read across is applied for this substance to fulfil these relevant endpoints.

Under the conditions of the present study C7-9-alkyl 3-(3,5-di-trans-butyl-4-hydroxyphenyl)propionate did not caused mortality of Eisenia fetida fetida at 1000 mg/kg d.s after 14 days of exposure (NOEC).

The 14-day LC₅₀was beyond the range tested, i.e. exceeded the limit concentration of 1000 mg/kg d.s.

 

The 14-day EC₅₀for weight reduction was beyond the range tested, i.e. exceeded the limit concentration of 1000 mg/kg d.s.

No classification is applicable.

Description of key information

Acute toxicity to earthworms.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Additional information

Given that it is recommended as RMM that CAS1879-09-0 is not released to the environment, this use of read across is considered to be an appropriate way to address the terrestrial endpoint. As such, read across is applied for this substance to fulfil these relevant endpoints.