4-(1,1,3,3-tetramethylbutyl)phenol

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods, other

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

In accordance with Regulation (EC) 1907/2006 Annex XI (1.5) and the relevant ECHA guidance documents, the substances detailed in the table below are grouped for the purposes of read across to reduce the need for unnecessary repeat testing on the basis that the substances are similar on the basis of a common functional groups.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Duration:

4 wk

Dose descriptor:

EC10

Effect conc.:

24 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (5.2, 110.7)

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

55.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mean number of live juvenile earthworms (offspring)

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

88.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Conclusions:

The read across for 4-tert-octylphenol (CAS: 140-66-9); is based upon the analogous substances to which basic form, degree of substitution of functional groups is not considered to effect the proposed read across for the endpoint of Toxicity to soil macroorganisms except arthropods. Based on the information available for the read across substances, the substance is expected to be toxic to soil macroorganisms except arthropods .

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2001-2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The document is a report to the UK Government and differs in style and content from a laboratory study report and a peer-reviewed publication. However, it contains full details on the methods used in the studies that support the paper, and its only issue appears to be that it does not state whether these are conducted to GLP. Therefore, it has been assigned a KIlimisch score of 2.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

ISO 11268-1 (Effects of Pollutants on Earthworms. 1. Determination of Acute Toxicity Using Artificial Soil Substrate)

Deviations:

yes

Remarks:

Slight deviation of standard temperature but this is not expected to affect the results.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

no

Details on sampling:

- Start of Test: Samples of soil were collected from each vessel at commencement of testing and stored at -20 °C prior to analysis.
- End of Test: Live worms were snap frozen in liquid nitrogen, then stored at -80 °C prior to analysis.

Vehicle:

yes

Details on preparation and application of test substrate:

- Vehicle: Acetone
- Preparation: For each exposure concentration a stock solution of the test substance was prepared in acetone and added to a quantity of soil such that the maximum volume of acetone used was 3.2 mg/kg. After spiking the soil, 0.5 kg was added to replicate test vessels and then left overnight to allow any remaining acetone to volatilise before adding the test animals.
- Controls: Negative control and solvent (acetone) controls were included in the study design.

Test organisms (species):

Eisenia sp.

Animal group:

annelids

Details on test organisms:

- Supplier: Earthworms were purchased from a commercial supplier (details not available).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Post exposure observation period:

Surviving earthworms were retained for biomarker analysis in relation to endocrine disruption studies.

Test temperature:

25 ± 2 °C

pH:

6.0 ± 0.5 (in accordance with the OECD Artificial Soil)

Moisture:

25 to 42% of the dry weight soil (in accordance with the OECD Artificial Soil)

Details on test conditions:

- Exposure: After spiking the soils, 0.5 kg of soil was added to each test vessel and left overnight for the acetone to volatilise before the earthworms were added to the test vessels.
- Test vessel: Kilner jars (glass).
- Number of organisms per concentration at the start of the test: 20 in control and solvent control; 10 in all test substance concentrations.
- Acclimation: Earthworms were added to each replicate and allowed to burrow into the soil.
- Food: 5 g of dried ground rabbit's droppings were placed on the surface of the soil in each vessel for the worms to feed on.
- Photoperiod: 16 hours light and 8 hours dark cycle.
- Weight of vessels: The weights of the vessels were recorded twice weekly so that weights could be adjusted to compensate for evaporative losses by the addition of distilled water.
- Removal: On day 14, live and dead adult earthworms were removed from the test vessels by sieving.

Nominal and measured concentrations:

Nominal Concentrations: 1.0, 3.2, 10, 32, 100, 320, 1000, 3200, 10000 mg/kg.

Reference substance (positive control):

no

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

88.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Quality Control: QC criteria were met with 0% lethality in the negative control and 10% lethality in the solvent control.

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

Statistics were used but the method is not reported. ENVIRON UK Ltd has statistically derived an LC50 using Probit analysis assuming up to 3 mortalities per control (i.e., less than 10% mortality). The statistical tool used was ProStat Ver. 5.01 for Windows by Poly Software International, Inc., P.O. Box 60, Pearl River, NY 10965 USA. The wide 95% confidence intervals seemed to be a function of control mortality.

The LC₅₀ is prioritised for reporting from this study as it a short-term exposure and directly comparable with the EC₅₀ from other short-term toxicity studies (e.g., Hulzebos et al, 1993). A NOEC and a LOEC based on growth effects of 32 and 100 mg nonylphenol/kg were also reported in this study.

Validity criteria fulfilled:

yes

Conclusions:

Nonylphenol was found to affect the survival of adult earthworms (Eisenia sp.) with a NOEC of 32 mg/kg and a LOEC of 100 mg/kg dry weight soil. This study was a precursor to further studies on the long-term effects of nonylphenol and other endocrine disrupting substances to earthworms and the aquatic amphipod Gammarus pulex. Further endpoints studied were reproduction and three genetic biomarkers (vitellogenin, a sperm marker and gene expression of annetocin). It was concluded that although earthworms are important test species for assessing the effects of substances to terrestrial ecosystems, the hermaphroditic nature of earthworms means that they are not appropriate for the assessment of endocrine disrupting effects.

Executive summary:

In a 14-day acute toxicity study, earthworms (Eisenia andreii) were exposed at control, solvent control, and nonylphenol at 1.0, 3.2, 10, 32, 100, 320, 1000, 3200, 10000 mg nonylphenol/kg artificial soil. The NOEC based on lethality was 32 mg nonylphenol/kg, with a LOEC of 100 mg nonylphenol/kg, respectively. ENVIRON derived a statistical LC₅₀ of 88.6 mg nonylphenol/kg which is taken as the key value for assessment. Based on the results of this study, nonylphenol would be considered to have adverse acute effects to Eisenia andreii.

This study is classified as acceptable and is similar in methodology to the requirements of the ISO Guideline 11268 -2; Effects of Pollutants on Earthworms (Part 2, Determination of Effects on Reproduction).

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The document is a report to the UK Government and differs in style and content from a laboratory study report and a peer-reviewed publication. However, it contains full details on the methods used in the studies that support the paper, and its only issue appears to be that it does not state whether these are conducted to GLP. Therefore, it has been assigned a KIlimisch score of 2.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Deviations:

yes

Remarks:

Slight deviation of standard temperature but this is not expected to affect the results.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

no

Details on sampling:

- Start of Test: Samples of soil were collected from each vessel at commencement of testing and stored at -20 °C prior to analysis.
- End of Test: Live worms were snap frozen in liquid nitrogen, then stored at -80 °C prior to analysis.

Vehicle:

yes

Details on preparation and application of test substrate:

- Vehicle: Acetone
- Preparation: For each exposure concentration a stock solution of the test substance was prepared in acetone and added to a quantity of soil such that the maximum volume of acetone used was 3.2 mg/kg. After spiking the soil, 0.5 kg was added to replicate test vessels and then left overnight to allow any remaining acetone to volatilise before adding the test animals.
- Controls: Negative control and solvent (acetone) controls were included in the study design.

Test organisms (species):

Eisenia sp.

Animal group:

annelids

Details on test organisms:

- Supplier: Earthworms were purchased from a commercial supplier (details not available).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Post exposure observation period:

Surviving earthworms were retained for biomarker analysis in relation to endocrine disruption studies.

Test temperature:

25 ± 2 °C

pH:

6.0 ± 0.5 (in accordance with the OECD Artificial Soil)

Moisture:

25 to 42% of the dry weight soil (in accordance with the OECD Artificial Soil)

Details on test conditions:

- Exposure: After spiking the soils, 0.5 kg of soil was added to each test vessel and left overnight for the acetone to volatilise before the earthworms were added to the test vessels.
- Test vessel: Kilner jars (glass).
- Number of organisms per concentration at the start of the test: 40 in control and solvent control; 40 in all test substance concentrations.
- Acclimation: Earthworms were added to each replicate and allowed to burrow into the soil.
- Food: 5g of dried ground rabbit's droppings were placed on the surface of the soil in each vessel for the worms to feed on.
- Photoperiod: 16 hours light and 8 hours dark cycle.
- Weight of vessels: The weights of the vessels were recorded twice weekly so that weights could be adjusted to compensate for evaporative losses by the addition of distilled water.
- Removal: On day 28, adult worms were removed from the test vessels by sieving then snap frozen in liquid nitrogen for later analysis. Sieved soil containing cocoons was returned to the vessels and left for a further 28 days to allow cocoons to hatch. At the end of the test (56 days) the number of juvenile worms in each vessel was counted after wet-sieving.

Nominal and measured concentrations:

Nominal Concentrations: 1.0, 3.2, 10, 32, 100 mg/kg.

Reference substance (positive control):

no

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

100 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mean number of live juvenile earthworms (offspring)

Details on results:

- Quality Control: QC criteria were met. Mean number of live juvenile worms at day 56 in negative control was 27.6 ± 14.6 (standard deviation) and in the solvent control was 28.9 ± 20.7. The mean number of live juvenile worms at day 56 in the highest concentration tested (100 mg/kg) was 23.0 ± 10.0 (standard deviation).

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

Statistics were used but the method is not reported.

A summary of the physiological effects measured in the long-term Eisenia andreii reproduction study is provided below:

Substance	Exposure concentration	Mean number of hatched cocoons	Mean number of unhatched cocoons	Mean number of live worms
Nonylphenol	Control	12.5+2.1	2.8+1.3	24.3+8.1
	1.0 mg kg^-1	13.5+4.4	3.0+1.4	23.0+10.6
	3.2 mg kg^-1	9.8+4.3	2.0+2.8	21.5+7.5
	10 mg kg^-1	13.3+2.5	3.0+1.6	22.5+4.7
	32 mg kg^-1	7.8+3.2	4.3+2.9	17.0+7.3
	100 mg kg^-1	11.0+5.3	2.3+1.7	23.0+10.0

Validity criteria fulfilled:

yes

Conclusions:

Nonylphenol was not found to affect the reproduction of adult earthworms (Eisenia sp.) with a NOEC of 100 mg/kg dry weight soil. This study was part of a broader study of the long-term effects of nonylphenol and other endocrine disrupting substances to earthworms and the aquatic amphipod Gammarus pulex. Further endpoints studied included three genetic biomarkers (vitellogenin, a sperm marker and gene expression of of annetocin). It was concluded that although earthworms are important test species for assessing the effects of substances to terrestrial ecosystems, the hermaphroditic nature of earthworms means that they are not appropriate for the assessment of endocrine disrupting effects.

Executive summary:

In an eight week chronic toxicity study, earthworms (Eisenia andreii) were exposed at control, solvent control, and nonylphenol at 1.0, 3.2, 10, 32, 100 mg nonylphenol/kg artificial soil. The NOEC based on reproduction was 100 mg nonylphenol/kg. Based on the results of this study, nonylphenol would not be considered to have adverse reproductive effects to Eisenia andreii.

This study is classified as acceptable and is similar in methodology to the requirements of the ISO Guideline 11268 -2; Effects of Pollutants on Earthworms (Part 2, Determination of Effects on Reproduction).

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Spring 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Published paper which meets international standard methods including OECD and ISO Guidelines.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: ISO Guideline 16387 Soil Quality - effects of pollutants on Enchytraeidae (Enchytraeus sp.) - determination of effects on survival and reproduction. Guideline No. 16387. International Organisation for Standardisation, Geneva, pp 1-22 (ISO 2003)

Deviations:

yes

Remarks:

The test vessels and volumes differed from the ISO protocol but this is not thought to have affected the results of the experiment. In addition, the test period was shortened in accordance with Kuperman et al (2006) as E. crypticus has a shorter generatio

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

no

Details on sampling:

No data

Vehicle:

yes

Details on preparation and application of test substrate:

- Vehicle: Nonylphenol was applied to soils dissolved in acetone (95%, Panreac, Barcelona, Spain).
- Application: To prepare the concentrations of test substance, the amount of soil to be used for each concentration was moistened with a fixed volume of acetone solution containing the appropriate quantity of nonylphenol. Acetone was then left to evaporate for 24 hours in a fume hood.
- Controls: Two control soils were prepared; one negative control and one solvent control to which the same volume of acetone was added as was used in the test substance concentrations.
- Concentrations: The test concentrations were selected following a preliminary assay consisting of 0, 1, 10, 100, 1000 and 10000 mg of nonylphenol per kg of soil. The range of concentrations showing an inhibition between 10% and 90% were selected for the study.

Test organisms (species):

other: Enchytraeus crypticus

Animal group:

annelids

Details on test organisms:

No further data on test organisms is provided.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

4 wk

Remarks:

This is a shorter exposure period than the standard 6-week duration in the ISO Protocol. However, the modifications were made because the enchytraeid species is more easily damaged and has a shorter generation time than E. albidus.

Post exposure observation period:

No data

Test temperature:

21 °C

pH:

6 ± 0.5 (as per OECD Artificial Soil) adjusted using calcium carbonate.

Moisture:

Water holding capacity: 44.8%
Moisture content: Soil water content was adjusted to 60% of the maximum water holding capacity.

Details on test conditions:

TEST SYSTEM
- OECD artificial soil: Prepared according to the OECD protocol for artificial soil (1984) by mixing Sphagnum peat (10%), kaoline (20%) and quartz sand.
- Testing facility: Universitat Autonoma de Barcelona
- Test container (type, material, size): Each replicate consisted of a 150-ml polyethylene flask.
- Amount of soil: Each vessel was filled with 30 g (dry weight)
- No. of replicates per treatment group: 5 replicates per concentration were prepared
- Number of organisms: Ten adults (clearly identified by the clitella were introduced into each flask.
- Feeding: Animals were fed with 25 mg of ground oats at the start and weekly thereafter.
- Photoperiod: Test was maintained in the dark.
- Aeration: Animals were aerated twice a week.

SOURCE AND PROPERTIES OF SUBSTRATE
- Geographic location: The results presented here relate to the OECD Artificial Soil, however two natural soils were also tested. These correspond to a loamy agricultural soil (PRA) within the Universitat Autonoma de Barcelona campus (Cerdanyola del Valles, NE Spain) and the other was a loam sand soil (UAB) from the dry grassland in Serra de Prades (Tarragona, NE Spain).
- Pesticide use history at the collection site: Not relevant to the OECD Artificial Soil, however, pesticide use had ceased five years previously in the agricultural soil (UAB).
- Collection procedures: Natural soils were defaunated by alternating two consecutive freeze-thaw cycles (-20 °C for 4 days, then +20 °C for 4 days).
- Sampling depth (cm): Not relevant to the OECD soil, but natural soils were collected from the upper layer (20 cm).
- Soil texture (if natural soil): PRA soil
- % sand: 80.3
- % silt: 16.5
- % clay: 3.2

- Soil texture (if natural soil): UAB soil
- % sand: 36.4
- % silt: 44.9
- % clay: 18.7

- Composition (if artificial substrate): OECD Artificial Soil
- Organic carbon (%): 3.45
- Maximum water holding capacity (in % dry weight): 44.8% adjusted to 60% WHC
- CEC: 8.94

END OF TEST
- Adults and juveniles were fixed by direct addition of ethanol (80% v/v) to each flask.
- A few drops of Bengal red (1% solution in ethanol) were added to colour the individuals. After 2 hours, individuals were separated from the more fine soil particles by rinsing soil with tap water in a sieve (0.2 mm) and then transferred to a Petri dish for counting.
- All the individuals counted were assumed to be alive given the quick degradation time of individuals once dead.

METHOD VARIATION
- The test was shortened from 6 weeks to 4 weeks in accoradance with Kuperman et al (2006). The full reference is Kuperman RG, Checkai RT, Simini M, Phillips CT, Anthony JS, Kolakowski JE and Davis EA (2006) Toxicity of emerging energetic soil contaminant CL-20 to potworm Enchytraeus crypticus in freshly amended or weathered and aged treatments. Chemosphere 62: 1282-1293.

Nominal and measured concentrations:

Nominal concentrations: The test concentrations were selected following a preliminary assay consisting of 0, 1, 10, 100, 1000 and 10000 mg of nonylphenol per kg of soil. The range of concentrations showing an inhibition between 10% and 90% were selected for the study.

Reference substance (positive control):

no

Duration:

4 wk

Dose descriptor:

EC10

Effect conc.:

24 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: (5.2, 110.7)

Details on results:

No differences in the outcomes between controls with and without the addition of acetone were found in any of the assays. Validity criteria were met for all species.

- Survival was over 80%
- Reproduction was over 25 juveiles with a coefficient of variation below 50%.

Mean values (+/- standard deviation) are provided for the controls in the different test soils below:
- Survival in adults: OECD 9.0 (±1.8); PRA 7.8 (±3.0); UAB 8.8 (±0.9)
- Reproduction in juveniles: OECD 391.8 (±179.6); PRA 702.0 (±322.8); UAB 777.2 (±311.3)

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

- EC10 was calculated using Statistica 6.0 (StatSoft Inc., Tulsa USA).
- The influence of soil properties was assessed by Generalised Linear Models (Brodgar 2.0, Highland Statistics Ltd, Newburgh, UK).

Reproduction was a more sensitive endpoint than survival in the encytraeid study. By way of comparison using the OECD artificial soil, the EC₁₀ for reproduction was 24.0 mg/kg compared with the EC₁₀ and EC₅₀ for survival which were 663.5 and 906.7 mg/kg respectively. When the results for the three soils were compared, enchytraeids (reproduction) in OECD Artificial Soil (EC₁₀ of 24.0 mg/kg) were more sensitive than enchytraeids in the other soils; reproduction in the PVA soil (EC₁₀ of 455.8 mg/kg) and UAB soil (

EC₁₀ of 197.2 mg/kg). The results for the OECD Artificial Soil have been selected for this hazard assessment as the standard soil data are directly comparable with other studies where artificial soils were used (for example, Johnson et al, 2005).

Toxicity values of 4 -nonylphenol for several terrestrial organisms in the three different soil types:

Species	Endpoint	Soil	EC10	EC50
B. rapa	Freshweight	OECD	574.8 (279.9, 1,180.4)	1,449.1 (785.0, 2,674.7)
	Germination		695.9 (286.3, 1,691.6)	8,159.2 (5,904.0, 11,276.0)
L. perenne	Freshweight	OECD	738.9 (49.6, 11,011.2)	4,011.6 (862.2, 18,664.5)
	Germination		1,385.8 (696.5, 2,757.3)	7,500.7 (6,031.2, 9,328.3)
E. andreii	Survival	OECD	343.9 (160.6, 736.4)	625.5 (427.9, 914.1)
		PRA	182.4 (157.6, 211.2)	290.7 (272.0, 310.7)
		UAB	498.2 (439.2, 565.2)	622.0 (592.3, 653.2)
	Reproduction	OECD	55.8 (13.7, 227.7)	82.0 (47.6, 141.1)
		PRA	43.1 (26.9, 69.0)	69.6 (57.7, 83.9)
		UAB	63.6 (46.5, 87.1)	124.3 (110.5, 139.9)
	Biomass	OECD	88.3 (50.0, 155.8)	309.1 (240.4, 397.3)
		PRA	121.2 (93.6, 156.9)	240.2 (218.0, 264.8)
		UAB	289.4 (219.1, 382.4)	523.2 (474.5, 576.9)
E. crypticus	Survival	OECD	663.5 (444.9, 989.5)	906.7 (786.5, 1,045.3)
		PRA	214.5 (171.3, 268.6)	316.2 (284.9, 350.9)
		UAB	227.0 (89.5, 575.3)	615.2 (431.9, 876.3)
	Reproduction	OECD	24.0 (5.2, 110.7)	225.8 (134.8, 378.4)
		PRA	455.8 (311.2, 667.5)	641.7 (554.2, 742.9)
		UAB	197.2 (97.9, 397.3)	212.8 (194.6, 232.8)
F. candida	Survival	OECD	102.0 (69.7, 149.3)	138.7 (129.4, 148.7)
		PRA	52.3 (47.0, 58.1)	69.8 (65.8, 74.1)
		UAB	89.1 (70.0, 113.5)	116.3 (104.2, 129.8)
	Reproduction	OECD	63.2 (47.3, 84.5)	92.8 (83.8, 102.8)
		PRA	45.0 (27.5, 73.7)	64.4 (54.6, 75.9)
		UAB	54.8 (23.9, 125.7)	88.0 (79.9, 96.9)

Validity criteria fulfilled:

yes

Conclusions:

The authors considered nonylphenol to be more toxic than the nonylphenol ethoxylate mixture and considered the results to be in agreement with existing published literature. Nonylphenol presented low phytotoxicity but moderate toxicity for invertebrates, reproduction being clearly the most sensitive endpoint. Nonylphenol generally affected the invertebrates' reproduction at lower concentrations than those affecting growth or survival. Earthworms and collembolans were more sensitive taxa compared to enchytraeids.

Executive summary:

In a 4-week chronic toxicity study, enchytraeids (Enchytraeus crypticus) were exposed at control, solvent control, and nonylphenol at a range of concentrations showing inhibition between 10% and 90% from an initial range-finding test. The EC₁₀ based on reproduction was 24 mg nonylphenol/kg, using an artificial soil test medium. Results for two natural soils were also reported. Based on the results of this study, nonylphenol would be considered to have adverse chronic and acute effects to Enchytraeus crypticus.

This study is classified as acceptable and is similar in methodology to the requirements of the ISO Guideline 16387 (ISO, 2003).

Reference 5

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Spring 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Published paper which meets international standard methods including OECD and ISO Guidelines.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)

Deviations:

no

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

no

Details on sampling:

No data

Vehicle:

yes

Details on preparation and application of test substrate:

- Vehicle: Nonylphenol was applied to soils dissolved in acetone (95%, Panreac, Barcelona, Spain).
- Application: To prepare the concentrations of test substance, the amount of soil to be used for each concentration was moistened with a fixed volume of acetone solution containing the appropriate quantity of nonylphenol. Acetone was then left to evaporate for 24 hours in a fume hood.
- Controls: Two control soils were prepared; one negative control and one solvent control to which the same volume of acetone was added as was used in the test substance concentrations.
- Concentrations: The test concentrations were selected following a preliminary assay consisting of 0, 1, 10, 100, 1000 and 10000 mg of nonylphenol per kg of soil. The range of concentrations showing an inhibition between 10% and 90% were selected for the study.

Test organisms (species):

Eisenia sp.

Animal group:

annelids

Details on test organisms:

No further data on test organisms is provided.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

8 wk

Post exposure observation period:

No data

Test temperature:

21 °C

pH:

6 ± 0.5 (as per OECD Artificial Soil) adjusted using calcium carbonate.

Moisture:

Water holding capacity: 44.8%
Moisture content: Soil water content was adjusted to 60% of the maximum water holding capacity.

Details on test conditions:

TEST SYSTEM
- OECD artificial soil: Prepared according to the OECD protocol for artificial soil (1984) by mixing Sphagnum peat (10%), kaoline (20%) and quartz sand.
- Testing facility: Universitat Autonoma de Barcelona
- Test container (type, material, size): Each replicate consisted of a 1000-ml polyethylene container covered with a perforated lid allowing aeration of the test chamber.
- Amount of soil: Each vessel was filled with 500g (dry weight) or moist soil.
- No. of replicates per treatment group: 4 replicates per concentration were prepared
- Number of organisms: Ten clitellated individuals of synchronised age (4 weeks difference at most) were placed in each container.
- Feeding: Animals were fed with 5 g cooked oat flakes at the start and weekly thereafter.
- Photoperiod: Test was maintained at 16: 8 light/dark photoperiod.
- Moisture loss: The vessels were weighed weekly to check for moisture loss and weight restored with distilled water where necessary.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: The results presented here relate to the OECD Artificial Soil, however two natural soils were also tested. These correspond to a loamy agricultural soil (PRA) within the Universitat Autonoma de Barcelona campus (Cerdanyola del Valles, NE Spain) and the other was a loam sand soil (UAB) from the dry grassland in Serra de Prades (Tarragona, NE Spain).
- Pesticide use history at the collection site: Not relevant to the OECD Artificial Soil, however, pesticide use had ceased five years previously in the agricultural soil (UAB).
- Collection procedures: Natural soils were defaunated by alternating two consecutive freeze-thaw cycles (-20 °C for 4 days, then +20 °C for 4 days).
- Sampling depth (cm): Not relevant to the OECD soil, but natural soils were collected from the upper layer (20 cm).
- Soil texture (if natural soil): PRA soil
- % sand: 80.3
- % silt: 16.5
- % clay: 3.2

- Soil texture (if natural soil): UAB soil
- % sand: 36.4
- % silt: 44.9
- % clay: 18.7

- Composition (if artificial substrate): OECD Artificial Soil
- Organic carbon (%): 3.45
- Maximum water holding capacity (in % dry weight): 44.8% adjusted to 60% WHC
- CEC: 8.94

Nominal and measured concentrations:

Nominal concentrations: The test concentrations were selected following a preliminary assay consisting of 0, 1, 10, 100, 1000 and 10000 mg of nonylphenol per kg of soil. The range of concentrations showing an inhibition between 10% and 90% were selected for the study.

Reference substance (positive control):

no

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

55.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

No differences in the outcomes between controls with and without the addition of acetone were found in any of the assays. Validity criteria were met for all species.

- Adult weight in worms at the start of the test was in the range inidicated in the Guideline (200-650 mg)
- Survival was over 90%
- Reproduction was over 30 juveiles with a coefficient of variation below 30%.

Mean values (+/- standard deviation) are provided for the controls in the different test soils below:
- Survival in adults: OECD 9.8 (±0.8); PRA 10.0 (±0.0); UAB 10.0 (±0.0)
- Reproduction in juveniles: OECD 53.4 (±14.6); PRA 73.1 (±16.2); UAB 104.2 (±26.7)
- Initial weight /worm: OECD 417 (±114); PRA 488 (±86); UAB 472 (±90)
- Final weight/worm: OECD 381 (±77); PRA 450 (±106); UAB 433 (±87)

Results with reference substance (positive control):

No data

Reported statistics and error estimates:

- EC10 was calculated using Statistica 6.0 (StatSoft Inc., Tulsa USA).
- The influence of soil properties was assessed by Generalised Linear Models (Brodgar 2.0, Highland Statistics Ltd, Newburgh, UK).

Reproduction was a more sensitive endpoint than survival in the earthworm study. By way of comparison using the OECD artificial soil, the EC₁₀ for reproduction was 55.8 mg/kg compared with the EC₁₀ and EC₅₀ for survival which were 343.9 and 625.5 mg/kg respectively. When the results for the three soils were compared, earthworms in PRA soil were more sensitive (EC₁₀ of 43.1 mg/kg) than the OECD Artificial Soil (EC₁₀ of 55.8 mg/kg) and UAB soil (EC₁₀ of 63.6 mg/kg). The results for the OECD Artificial Soil have been selected for this hazard assessment as the standard soil data are directly comparable with other studies where artificial soils were used (for example, Johnson et al, 2005).

Toxicity values of 4 -nonylphenol for several terrestrial organisms in the three different soil types:

Species	Endpoint	Soil	EC10	EC50
B. rapa	Freshweight	OECD	574.8 (279.9, 1,180.4)	1,449.1 (785.0, 2,674.7)
	Germination		695.9 (286.3, 1,691.6)	8,159.2 (5,904.0, 11,276.0)
L. perenne	Freshweight	OECD	738.9 (49.6, 11,011.2)	4,011.6 (862.2, 18,664.5)
	Germination		1,385.8 (696.5, 2,757.3)	7,500.7 (6,031.2, 9,328.3)
E. andreii	Survival	OECD	343.9 (160.6, 736.4)	625.5 (427.9, 914.1)
		PRA	182.4 (157.6, 211.2)	290.7 (272.0, 310.7)
		UAB	498.2 (439.2, 565.2)	622.0 (592.3, 653.2)
	Reproduction	OECD	55.8 (13.7, 227.7)	82.0 (47.6, 141.1)
		PRA	43.1 (26.9, 69.0)	69.6 (57.7, 83.9)
		UAB	63.6 (46.5, 87.1)	124.3 (110.5, 139.9)
	Biomass	OECD	88.3 (50.0, 155.8)	309.1 (240.4, 397.3)
		PRA	121.2 (93.6, 156.9)	240.2 (218.0, 264.8)
		UAB	289.4 (219.1, 382.4)	523.2 (474.5, 576.9)
E. crypticus	Survival	OECD	663.5 (444.9, 989.5)	906.7 (786.5, 1,045.3)
		PRA	214.5 (171.3, 268.6)	316.2 (284.9, 350.9)
		UAB	227.0 (89.5, 575.3)	615.2 (431.9, 876.3)
	Reproduction	OECD	24.0 (5.2, 110.7)	225.8 (134.8, 378.4)
		PRA	455.8 (311.2, 667.5)	641.7 (554.2, 742.9)
		UAB	197.2 (97.9, 397.3)	212.8 (194.6, 232.8)
F. candida	Survival	OECD	102.0 (69.7, 149.3)	138.7 (129.4, 148.7)
		PRA	52.3 (47.0, 58.1)	69.8 (65.8, 74.1)
		UAB	89.1 (70.0, 113.5)	116.3 (104.2, 129.8)
	Reproduction	OECD	63.2 (47.3, 84.5)	92.8 (83.8, 102.8)
		PRA	45.0 (27.5, 73.7)	64.4 (54.6, 75.9)
		UAB	54.8 (23.9, 125.7)	88.0 (79.9, 96.9)

Validity criteria fulfilled:

yes

Conclusions:

The authors considered nonylphenol to be more toxic than the nonylphenol ethoxylate mixture and considered the results to be in agreement with existing published literature. Nonylphenol presented low phytotoxicity but moderate toxicity for invertebrates, reproduction being clearly the most sensitive endpoint. Nonylphenol generally affected the invertebrates's reproduction at lower concentrations than those affecting growth or survival. Earthworms and collembolans were more sensitive taxa compared to enchytraeids.

Executive summary:

In an 8-week chronic toxicity study, earthworms (Eisenia andreii) were exposed at control, solvent control, and nonylphenol at a range of concentrations showing inhibition between 10% and 90% from an initial range-finding test. The EC₁₀ based on reproduction was 55.8 mg nonylphenol/kg, using an artificial soil test medium. Results for two natural soils were also reported. Based on the results of this study, nonylphenol would be considered to have adverse acute effects to Eisenia andreii.

This study is classified as acceptable and is similar in methodology to the requirements of the ISO Guideline 11268 -2 (ISO, 1998).

Description of key information

The Johnson et al (2005) short-term LC50 of 88.6 mg nonylphenol/kg  for Eisenia andreii survival meets reliability ownership and adequecy requirements for REACH.  The study uses the preferred test species and the preferred test medium, artificial soil.  The long-term study of Domene et al (2009) followed international OECD and ISO protocols, uses standard test species and achieves the highest Klimisch rating.  Long-term exposure of Enchytraeus crypticus reported an EC10 of 24 mg nonylphenol/kg.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

88.6 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

24 mg/kg soil dw

Additional information

Only one reliable study was available for short-term exposure to soil macro-organisms except arthropods and this was the Key Study by Johnson et al., (2005). The preferred earthworm species, Eisenia andreii was exposed to nonylphenol for 14 days with a reported NOEC and LOEC based on survival of 32 and 100 mg/kg, respectively. ENVIRON UK Ltd calculated an LC₅₀of 88.6 mg nonylphenol/kg for the survival data using Probit statistical analysis. The derivation of an LC₅₀means the macro-invertebrate data are more easily comparable with other short-term studies, for example plant study by Hulzeboset al., 1993), where macro-invertebrates appeared to be more sensitive than plants.

Two studies representing three endpoints were reported for long-term macro-invertebrate exposures to nonylphenol resulting in NOEC or EC10 values ranging from 24 to 100 mg nonylphenol/kg. Both Johnsonet al., 2005 and Domeneet al., 2009 carried out 8-week earthworm (Eisenia andreii) reproduction studies using nonylphenol spiked artificial soil exposures. The Johnsonet al., 2005 study did not record any effects on reproduction at the highest concentration tested (NOEC = 100 mg/kg) but the Domeneet al., 2009 study statistically derived a more robust EC₁₀of 55.8 mg/kg. Domeneet al., 2009 also studied reproduction inEnchytraeus crypticusand statistically derived an EC₁₀of 24 mg nonylphenol/kg. TheE. crypticus study duration was 4 weeks and slightly shorter in duration than the standard 6-week duration of the standard ISO Guideline but this is still considered to be acceptable as this smaller enchytraeid species’ breeding cycle was completed.

In addition, Domene et al., 2009 compared the standard toxicity tests between OECD artificial soils and two natural soils. In the earthworm tests where reproduction in the OECD was affected at the 10% level leading to an EC₁₀of 55.8 mg/kg, the loamy sand soil from dry grassland affected reproduction at 43.1 mg/kg (more sensitive) but the agricultural loamy soil was less sensitive with an EC₁₀of 63.6 mg/kg. However, when the enchytraeid study was compared, both natural soils reported far less sensitive values than the OECD soil; 455.8 and 197.2 mg/kg compared to the EC₁₀of 24 mg/kg in the OECD soil. The inconsistency in trends between natural and artificial soils means that the artificial soils are preferred for use in this REACH asseessment to ensure studies are comparable where possible.

The long-term study of Domene et al., (2009) forEnchytraeus crypticuswith an EC₁₀of 24 mg nonylphenol/kg is the Key Study for this endpoint and is supported by the less sensitive long-term and short-term earthworm studies.