2-butyloctanoic acid

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2019 - October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

study was conducted and reported in compliance with present OECD, EC and German principles of Good Laboratory Practice

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Version from 2016

Deviations:

yes

Remarks:

Deviations were considered to have no impact on quality and integrity of the study (for details see "Any other information of materials and methods incl. tables" below).

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP Certificate: Statement of GLP Compliance from Staatliches Gewerbeaufsichtsamt Hildesheim, Germany, dated 03.01.2017

Analytical monitoring:

yes

Details on sampling:

- Concentrations: pooled samples of control, vehicle control and all test item concentrations at test start and test end, additionally the highest test substance concentration in soil for every replicate at test start, the stock solutions (acetone) of every test concentrations at test start
- Sample storage conditions before analysis: All original samples were stored at room temperature before preparation. Prepared samples were stored in an autosampler at room temperature until analysis.

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

- Method of mixing into soil: The respective test item amount for each test item concentration was weighed out, dissolved in acetone (2.5 mL per replicate) and mixed thoroughly with quartz sand (10 g per replicate). After complete evaporation of the solvent, the spiked quartz sand was added to the artificial soil (2400 g sdw). Additional demineralised water (272 g) was added to adjust the humidity of the artificial soil to a moisture of 54 % of the WHCmax. Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 610 g soil dry weight were filled into each test vessel.
- Controls: artificial soil moistened with demineralised water without test or reference item was used as control medium, artificial soil, spiked with acetone and moistened with demineralised water, without test or reference item was used as solvent control medium
- 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
- Source: Breeding stock culture maintained at the test facility
- Age at test initiation: 2-12 months, difference of age did not deviate by more than 1 month (achieved by synchronisation of population: placing adult earthworms into breeding boxes and removing the adults after 4 weeks, offspring from the remaining cocoons reached the adult age after 2 months at the earliest)
- Weight at test initiation: 0.36 to 0.54 g

ACCLIMATION
- Acclimation period: 2 days
- Acclimation conditions: same as test
- Health during acclimation (any mortality observed): nothing mentioned

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

18 - 23 °C

pH:

5.98 - 6.27

Moisture:

20.3 - 24.6% of dry weight

Details on test conditions:

TEST SYSTEM
- Test container: round plastic boxes, inner diameter = 15.0 cm corresponding to bottom surface area of 177 cm2, height = 14 cm, with transparent and perforated lids
- Amount of soil or substrate: 610 g soil dry weight
- No. of organisms per container (treatment): 10 adult earthworms (with clitellum)
- No. of replicates per treatment group: 4
- No. of replicates per control: 8
- No. of replicates per vehicle control: 8

PROPERTIES OF SUBSTRATE)
- Composition of artificial substrate: 5 % peat, air-dried and finely ground, 20 % kaolin, kaolinite content > 30 %, 74 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm), 0.20 % calcium carbonate (CaCO3) to achieve a pH of 6.27
- Maximum water holding capacity (in % dry weigth): 38.9
- Pretreatment of soil: Two days before test start the artificial soil (27621 g SDW) was pre-moistened with 2621 g demineralised water to achieve a moisture content corresponding to 27 % of the WHCmax of the artificial soil.
- Storage (condition, duration): the moistened soil was kept covered to prevent evaporative water losses prior to the start of exposure
- Stability and homogeneity of test material in the medium: not determined

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 400 - 800 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Body weight of adult earthworms: determined individually on day 0 and day 28 for each replicate
- Mortality and morphological changes of adult earthworms: after the first 4 weeks the earthworms were removed and examined, effects recorded for each replicate
- Observation parameters and scoring system:
A) no obvious pathological symptoms
B) no reaction to touching
C) no negative phototactical reaction
D) spontaneous segmentation and separation
E) spasmodic winding
F) yellow excretion from the oral aperture
G) ulcer and skin bleedings
H) dead earthworms
- Number of offsprings (juveniles) hatched from the cocoons: counted for each test container at the end of the experimental phase
- pH value, moisture content: at experimental starting and experimental end in each treatment and the controls from pooled samples of all replicates
- Water content: each test container was checked weekly by weighing, weight loss was replenished with the appropriate amount of demineralised water

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: A 14-day range finding test was performed with 4 test item concentrations each containing 2 replicates with 10 earthworms each, observations on mortality and change of live weight of adult earthworms were made.
- Test concentrations: control, 1, 10, 100, 1000 mg/kg soil dw
- Results used to determine the conditions for the definitive study: At test item concentrations of 1 and 10 mg/kg soil dw not effects on mortality of adult earthworms were measured. At 100 mg/kg soil dw, 25% adult earthworms were dead, at 1000 mg/kg soil dw 100% mortality were found. Change of live weight of the earthworms was not affected at 1, 10, and 100 mg/kg soil dw, not determinable at 1000 m/kg soil dw due to 100% mortality at this test item concentration.

Nominal and measured concentrations:

Nominal: 3.75 – 7.5 – 15 – 30 – 60 – 120 mg test item/kg soil dry weight
Measured: see Table #1 an #2 in section "Any other information on materials and methods incl. tables" below

Reference substance (positive control):

yes

Key result

Duration:

8 wk

Dose descriptor:

EC10

Effect conc.:

43.4 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95 % Confidence Interval: 13.8 - > 120 mg/kg soil dw

Duration:

8 wk

Dose descriptor:

EC50

Effect conc.:

> 120 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

30 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 120 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

>= 120 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: biomass

Details on results:

- Mortality at end of exposure period: see tables #3 and #4 below in section "Any other information on results incl. tables"
- Total mass of adults at beginning of test: see tables #3 and #5 below in section "Any other information on results incl. tables"
- Changes in body weight of live adults (% of initial weight) at end of exposure period: no statistically significant differences in earthworm biomass at all test item concentrations compared to the pooled control, see tables #3 and #5 below in section "Any other information on results incl. tables"
- No. of offspring produced: reproduction rate (average number of juveniles produced) was 105 in the pooled control and ranged from 65 to 114 in the test item treatment rates, no statistically significant differences in earthworm reproduction in the treatment rates 3.75 to 30 mg test item/kg SDW compared to the pooled control, at the test item concentrations 60 and 120 mg test item/kg SDW the earthworm reproduction was statistically significantly reduced. For details see tables #3 and #6 below in section "Any other information on results incl. tables"
- No. of unhatched cocoons: not mentioned
- Morphological abnormalities: No significant pathological symptoms in the controls and all test item concentrations
- Behavioural abnormalities: No significant changes in the behaviour of adult earthworms in the controls and all test item concentrations
- Other biological observations: none

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: significant effect levels: 1, 2 and 4 mg Carbendazim/kg soil dw, according to OECD 222 significant effects of Carbendazim should be observed between 1 and 5 mg a.i./kg soil dw.
- Other: none

Reported statistics and error estimates:

- Mortality of Adult Earthworms after 28 Days:
Fisher`s Exact Binomial Test with survival at 4 wks: Two-sample comparisons between the two controls.
Threshold concentrations (NOEC) for Survival at 4 wks: Qualitative Trend Analysis by Contrasts (Monotonicity of Concentration/Response). Tarone´s test for extra-binomial variance, Step-down CochranArmitage test was performed.
- Growth of Adult Earthworms after 28 Days:
NOEC/LOEC determination: Shapiro-Wilk´s Test on Normal Distribution.
STUDENT-t test for Homogeneous Variances with weight at 4 wks: Two-sample comparison of the two controls.
Levene´s Test on Variance Homogeneity (with Residuals) with weight at 4 wks and the mediantest (2-by-2 table) with Bonferroni correction was performed.
- Earthworm Reproduction:
NOEC/LOEC determination: Comparison between Control and Solvent Control for Offspring Number at 8 wks by Shapiro-Wilk´s Test on Normal Distribution, STUDENT-t test for Homogeneous Variances with offspring number at 8 wks: Two-sample comparison of the two controls.
Threshold concentrations (NOEC) for Offspring Number at 8 wks: Shapiro-Wilk´s Test on Normal Distribution, Levene´s Test on Variance Homogeneity (with Residuals), Trend analysis by contrasts (monotonicity of concentration/response) with offspring number at 8 wks, comparison of treatments with "Pooled Control" by the t test procedure after Williams with offspring number at 8 wks was performed.
ECx determination: Analysis of Variance and Test for Lack of Fit for the 3-param. normal CDF with offspring number at 8 wks, Point estimates from the 3-param. normal CDF, confidence limits of the EC10 used as a parameter were computed by means of the standard error of parameter b1; confidence limits for the remaining ECx were estimated by Monte-Carlo simulation using the parameter errors obtained from the inverse Hessian matrix.

Table #3: Summary of All Observed Effects in the Application rates

Application rates [mg test item/kg soil dw]	3.75	7.5	15	30	60	120
Mortality of adult earthworms	No	No	No	No	No	No
Body weight gain	No	No	No	No	No	No
Reduced reproduction rate (Number of juveniles)	No	No	No	No	Yes	Yes
Summary of all effects	No	No	No	No	Yes	Yes

Table #4: Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate	Replicate
[mg test item/kg soil dw]	1	2	3	4	5	6	7	8	Mean
Control	30	10	0	0	0	0	10	0	6.25
Solvent control	0	0	0	20	0	0	0	0	2.5
Pooled control	-								4.38
3.75	0	0	70	0	-	-	-	-	17.5
7.5	0	10	0	0	-	-	-	-	2.5
15	10	10	10	0	-	-	-	-	7.5
30	0	10	10	10	-	-	-	-	7.5
60	0	0	10	20	-	-	-	-	7.5
120	10	20	0	0	-	-	-	-	7.5

Table #5: Body Weight Changes of the Adult Earthworms

Application rate	Live weight on day 0			Live weight on day 28			Mean body weight changes± SD		Sig.
[mg test item/kg soil dw]	sum of all replicates [g]	N	mean ± SD (per replicate) [g]	sum of all replicates [g]	N	mean ± SD (per replicate) [g]	[g]	[%]
Control	35.9	80	0.45 ± 0.04	38.7	75	0.52 ± 0.08	0.07 ± 0.03	15.0 ± 6.46	-
Solvent control	35.9	80	0.45 ± 0.04	41.1	78	0.53 ± 0.06	0.08 ± 0.01	17.6 ± 3.20	-
Pooled contol	-	-	-	-	-	-	0.07 ± 0.02	16.3 ± 5.10	-
3.75	18.0	40	0.45 ± 0.05	18.0	33	0.54 ± 0.08	0.09 ± 0.03	20.4 ± 6.10	No
7.5	18.0	40	0.45 ± 0.05	20.8	39	0.53 ± 0.09	0.08 ± 0.03	17.8 ± 7.46	No
15	17.9	40	0.45 ± 0.04	19.3	37	0.52 ± 0.07	0.07 ± 0.01	16.2 ± 2.02	No
30	18.0	40	0.45 ± 0.05	19.6	37	0.53 ± 0.06	0.08 ± 0.02	18.4 ± 3.79	No
60	18.1	40	0.45 ± 0.04	19.7	37	0.53 ± 0.07	0.08 ± 0.03	17.7 ± 7.36	No
120	18.1	40	0.45 ± 0.05	21.2	37	0.57 ± 0.09	0.12 ± 0.03	27.3 ± 7.35	No

N = Number of earthworms, SD = standard deviation, Sig. = Statistical significance compared to control (Multiple Sequentially-rejective Median (2x2 -Table) Test after Bonferroni-Holm, α = 0.05)

Table #6: Reproduction Rate (Number of Juveniles after 8 Weeks)

Application rate [mg test item/kg soil dry weight]	Number of juveniles mean ± SD	CV [%]	% of control	Sig.
Control	95 ± 21.2	22.2	-	-
Solvent control	115 ± 29.2	24.4	-	-
Pooled control	105 ± 26.6	25.4	-	-
3.75	91 ± 25.2	27.7	86.7	No
7.5	101 ± 10.1	10.0	96.2	No
15	114 ± 35.7	31.3	109	No
30	107 ± 6.70	6.26	102	No
60	77 ± 13.1	17.0	73.3	Yes
120	65 ± 15.2	23.4	61.9	Yes

SD = Standard deviation, CV = Coefficient of variation, Sig. = Statistical significance compared to control (William's Multiple Sequential t-test Procedure, α = 0.05)

Validity criteria fulfilled:

yes

Conclusions:

Neither evident earthworm mortalities (< 10%) nor evident pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and the test item concentrations 7.5 to 120 mg test item/kg SDW. However, at the test item concentration 3.75 mg/kg 17.5 % mortality (not statistically significant) was observed. Since no statistically significant difference was observed, the control and the solvent control were pooled for further evaluation. There were no statistically significant differences in earthworm biomass at all test item concentrations compared to the pooled control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 105 in the pooled control and ranged from 65 to 114 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 3.75 to 30 mg test item/kg SDW compared to the pooled control. However, at the test item concentrations 60 and 120 mg test item/kg SDW the earthworm reproduction was statistically significantly reduced. The following EC, LOEC, NOEC-values were calculated: NOEC (mortality) ≥ 120 mg/kg soil dw, LOEC (mortality) > 120 m/kg soil dw, NOEC (reproduction) = 30 mg/kg soil dw, LOEC (reproduction) = 60 mg/kg soil dw, EC10 (reproduction) = 43.4 mg/kg soil dw (95% confidence interval: 13.8 - > 120 mg/kg soil dw), EC20 (reproduction) = 66.7 mg/kg soil dw (95% confidence interval: 19.7 - > 120 mg/kg soil dw), EC50 (reproduction) > 120 mg/kg soil dw.

Executive summary:

Effects of 2-butyloctancoic acid on mortality, biomass and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD 222 (2016) and in compliance with GLP. The study was conducted under static conditions over 8 weeks with the test item concentrations 3.75 – 7.5 – 15 – 30 – 60 – 120 mg test item/kg. Each application rate was mixed into artificial soil containing 5 % peat. A control and a solvent control including untreated artificial soil and untreated artificial soil spiked with acetone, respectively, were tested under the same conditions as the test item treatments. 80 test organisms were inserted into 8 control replicates, each. 40 test organisms were divided into 4 replicates for each test item treatment (10 earthworms per replicate). They had an individual body weight between 0.36 and 0.54 g at experimental starting. The amount of test item in soil was analysed via LC-MS/MS in all test item concentrations and controls on day 0 using pooled samples of all replicates per concentration level. Additionally, the amount of test item in soil of the highest tested concentration for every replicate as well as the stock solutions of every test item concentration were analytically verified at test start. The measured concentrations in soil were within the range of 92 to 114 % of the nominal values on day 0, demonstrating the right preparation of the tested concentrations. After 28 days of exposure in soil, neither evident earthworm mortalities (< 10%) nor evident pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and the test item concentrations 7.5 to 120 mg test item/kg SDW. However, at the test item concentration 3.75 mg/kg 17.5 % mortality (not statistically significant) was observed. The control and the solvent control were pooled for further evaluation, since no statistically significant difference was observed. There were no statistically significant differences in earthworm biomass at all test item concentrations compared to the pooled control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 105 in the pooled control and ranged from 65 to 114 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 3.75 to 30 mg test item/kg SDW compared to the pooled control. However, at the test item concentrations 60 and 120 mg test item/kg SDW the earthworm reproduction was statistically significantly reduced. All validity criteria recommended by the test guidelines were fulfilled.

The following EC, LOEC, NOEC-values were calculated:

NOEC (mortality) ≥ 120 mg/kg soil dw,
LOEC (mortality) > 120 mg/kg soil dw,

NOEC (reproduction) = 30 mg/kg soil dw,

LOEC (reproduction) = 60 mg/kg soil dw,

EC10 (reproduction) =  43.4 mg/kg soil dw (95% confidence interval: 13.8 - > 120 mg/kg soil dw),

EC20 (reproduction) =  66.7 mg/kg soil dw (95% confidence interval: 19.7 - > 120 mg/kg soil dw),

EC50 (reproduction) > 120 mg/kg soil dw.

Description of key information

The following EC, LOEC, NOEC-values were calculated from an Earthworm Reproduction Study according to OECD 222: NOEC (mortality) ≥ 120 mg/kg soil dw, LOEC (mortality) > 120 m/kg soil dw, NOEC (reproduction) = 30 mg/kg soil dw, LOEC (reproduction) = 60 mg/kg soil dw, EC10 (reproduction) =  43.4 mg/kg soil dw, EC20 (reproduction) =  66.7 mg/kg soil dw, EC50 (reproduction) > 120 mg/kg soil dw.

Key value for chemical safety assessment

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

43.4 mg/kg soil dw

Additional information

Neither evident earthworm mortalities (< 10%) nor evident pathological symptoms or changes in the behaviour of adult earthworms were observed in the control and the test item concentrations 7.5 to 120 mg test item/kg SDW. There were no statistically significant differences in earthworm biomass at all test item concentrations compared to the pooled control. After a further four weeks, the reproduction rate (average number of juveniles produced) was 105 in the pooled control and ranged from 65 to 114 in the test item treatment rates. There were no statistically significant differences in earthworm reproduction in the treatment rates 3.75 to 30 mg test item/kg SDW compared to the pooled control. However, at the test item concentrations 60 and 120 mg test item/kg SDW the earthworm reproduction was statistically significantly reduced. The following EC10 value was calculated: EC10 (reproduction) =  43.4 mg/kg soil dw (95% confidence interval: 13.8 - > 120 mg/kg soil dw).