Alkanes, C16-47, branched and linear

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 other: mg/kg

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL not stated

Key result

Duration:

14 d

Dose descriptor:

NOEC

Effect conc.:

1 000 other: mg/kg

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CL not stated

Details on results:

Range-finding Test
Cumulative mortality data from the exposure of Eisenia foetida to the test item during the range-finding test are given in Table 1 (see in any other information on results).
The results showed no mortalities at any of the test concentrations employed in the range-finding test.
Based on this information, a single test concentration of six replicates, at 1000 mg/kg was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that at the maximum test concentration given in the OECD/EEC test guidelines no mortalities or sub-lethal effects of exposure were observed.
Definitive Test
Mortality data
Cumulative mortality data from the exposure of Eisenia foetida to the test item during the definitive test are given in Table 2 (see in any other information on results).
Inspection of the mortality data based on the nominal test concentrations gave the following results:
Time (days) LC50 (mg/kg)
7 > 1000
14 > 1000
There were no mortalities or sub-lethal effects of exposure in 60 earthworms exposed to a test concentration of 1000 mg/kg for a period of 14 days.
It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg/kg.
There were no observed sub-lethal effects of exposure of Eisenia foetida to the test item.
Earthworm weight data
Statistical analysis using Bartlett's test for homogeneity of variance (Sokal and Rohlf 1981) and a Students t-test was carried out on the earthworm weight data (see Table 3 and Appendix 1 and Appendix 2) see in any other information on results.
There were significant differences (P>0.05) between the control and 1000 mg/kg test groups in terms of weight on Day 0.
There were no significant differences (P>0.05) between the control and all the test groups in terms of weight on Day 14.
The worm weights for the control at Day 0 were found to be significantly heavier (P>0.05) than the 1000 mg/kg test group, however as there were no significant differences in terms of weight on Day 14 it was considered that the differences observed on Day 0 did not affect the outcome or validity of the test.
No observed effect concentration
The No Observed Effect Concentration (NOEC) was 1000 mg/kg on the basis that no mortalities were observed after 14 days exposure and additionally no sub-lethal effects on weight or behaviour were observed at 1000 mg/kg.
Physico-chemical measurements
The measured pH values and soil moisture content in the control and test cultures are given in Appendix 3 (see in any other information on results). There were no significant changes in pH or moisture content over the test period. pH values ranged from 6.1 to 6.3 at initiation and 6.3 to 6.5 at termination. The soil moisture contents were 29% to 30% at initiation and 25% at termination.

Results with reference substance (positive control):

Positive Control
Cumulative mortality data from the exposure of Eisenia foetida to chloroacetamide during the positive control are given in Table 4 (see in any other information on results section). The relationship between mortality and concentration after 7 and 14 days is given in Figures 1 and 2 (see attached section).
Analysis of the mortality data by the trimmed Spearman-Karber method (Hamilton et al 1977) on Days 7 and 14 gave the following results based on nominal test concentrations:
Time (days) LC50 (mg/kg) 95% Confidence limits (mg/kg)
7 44 42 – 45
14 43 41 – 45
The No Observed Effect Concentration was 18 mg/kg.
The results from the positive control with chloroacetamide are within the normal range for this reference item of 10 to 50 mg/kg.

Reported statistics and error estimates:

The LC50 values and associated confidence limits were calculated by the probit method (Finney 1971) on Day 7 and by the trimmed Spearman-Karber method (Hamilton et al 1977) on Day 14 using the ToxCalc computer software package (ToxCalc 1999).

Table 1: Cumulative Mortality Data in the Range-finding Test

Nominal Concentration (mg/kg)	Cumulative Mortality (Initial Population: 10)		% Mortality
	Day 7	Day 14	Day 7	Day 14
Control	0	0	0	0
10	0	0	0	0
100	0	0	0	0
1000	0	0	0	0

Table 2: Cumulative Mortality Data in the DefinitiveTest

Nominal Concentration (mg/kg)		Cumulative Mortality (Initial Population : 10)		% Mortality
		Day 7	Day 14	Day 7	Day 14
Control	R1	0	0	0	0
	R2	0	0	0	0
	R3	0	0	0	0
	R4	0	0	0	0
1000	R1	0	0	0	0
	R2	0	0	0	0
	R3	0	0	0	0
	R4	0	0	0	0
	R5	0	0	0	0
	R6	0	0	0	0

R₁– R₆= Replicates 1 to 6

 Table 3: Mean Earthworm Weights in the Definitive Test

Nominal Concentration (mg/kg)		Mean Earthworm Weight (g)
		Day 0		Day 14
		Mean	SD	Mean	SD
Control	R1	0.46	0.08	0.36	0.06
	R2	0.48	0.08	0.40	0.06
	R3	0.53	0.06	0.45	0.07
	R4	0.41	0.05	0.35	0.04
1000	R1	0.41	0.09	0.36	0.10
	R2	0.43	0.07	0.35	0.07
	R3	0.46	0.10	0.39	0.10
	R4	0.39	0.05	0.36	0.04
	R5	0.42	0.09	0.37	0.06
	R6	0.45	0.08	0.40	0.07

SD= Standard deviation

R₁– R₆= Replicates 1 to 6

 Appendix 1: Individual Worm Weights (g) – Day 0

Nominal Concentration (mg/kg)		Individual Worm Weights (g)
		1	2	3	4	5	6	7	8	9	10
Control	R1	0.53	0.60	0.58	0.42	0.39	0.40	0.37	0.40	0.50	0.44
	R2	0.56	0.50	0.37	0.39	0.52	0.42	0.51	0.41	0.55	0.57
	R3	0.60	0.53	0.43	0.57	0.50	0.60	0.53	0.54	0.45	0.59
	R4	0.49	0.37	0.42	0.37	0.40	0.38	0.44	0.39	0.49	0.38
1000	R1	0.40	0.42	0.37	0.59	0.35	0.30	0.38	0.42	0.34	0.55
	R2	0.38	0.39	0.39	0.45	0.53	0.54	0.42	0.39	0.30	0.48
	R3	0.54	0.39	0.47	0.38	0.47	0.31	0.38	0.45	0.59	0.60
	R4	0.40	0.42	0.34	0.39	0.48	0.38	0.35	0.36	0.31	0.44
	R5	0.35	0.49	0.30	0.42	0.39	0.55	0.37	0.38	0.56	0.40
	R6	0.33	0.49	0.38	0.52	0.42	0.58	0.47	0.38	0.48	0.40

R₁– R₆= Replicates 1 to 6

Appendix 2: Individual Worm Weights (g) – Day 14

Nominal Concentration (mg/kg)		Individual Worm Weights (g)
		1	2	3	4	5	6	7	8	9	10
Control	R1	0.36	0.39	0.32	0.31	0.33	0.30	0.37	0.47	0.44	0.35
	R2	0.44	0.38	0.50	0.38	0.37	0.39	0.46	0.31	0.33	0.43
	R3	0.49	0.48	0.53	0.56	0.46	0.38	0.36	0.40	0.40	0.43
	R4	0.40	0.31	0.37	0.36	0.34	0.30	0.39	0.39	0.30	0.31
1000	R1	0.35	0.50	0.55	0.30	0.41	0.30	0.30	0.33	0.30	0.27
	R2	0.27	0.31	0.49	0.44	0.28	0.31	0.36	0.41	0.32	0.30
	R3	0.52	0.40	0.36	0.36	0.40	0.30	0.31	0.40	0.59	0.28
	R4	0.38	0.36	0.38	0.29	0.37	0.39	0.29	0.41	0.35	0.35
	R5	0.45	0.32	0.32	0.44	0.36	0.34	0.36	0.46	0.29	0.36
	R6	0.34	0.41	0.47	0.40	0.42	0.34	0.52	0.34	0.33	0.46

R₁– R₆= Replicates 1 to 6

Appendix 3: Soil Moisture Content and pH Values

i)         Soil Moisture Content

Nominal Concentration (mg/kg)	Soil Moisture Content % (Expressed as % of Dry Weight)
	Day 0	Day 14
Control	29	25
1000	30	25

 

ii)        pH Values

Nominal Concentration (mg/kg)		Day 0	Day 14
Control	R1	6.3	6.3
	R2	6.3	6.3
	R3	6.4	6.3
	R4	6.3	6.3
1000	R1	6.2	6.5
	R2	6.2	6.4
	R3	6.2	6.3
	R4	6.2	6.3
	R5	6.2	6.3
	R6	6.1	6.3

R₁– R₆= Replicates 1 to 6

Table 4: Cumulative Mortality Data in the Positive Control with Chloroacetamide

Nominal Concentration (mg/kg)	Cumulative Mortality (Initial Population: 40)		% Mortality
	Day 7	Day 14	Day 7	Day 14
Control	0	0	0	0
5.6	0	0	0	0
10	0	0	0	0
18	0	0	0	0
32	0	1	0	2.5
56	36	36	90	90

The light intensity was 532 to 649 lux (see Appendix 4 see below).

Appendix 4: Room Temperature and Light Intensity Records

Day	Room Temperature (°C)		Light Intensity (Lux)
	Min	Max
0	22	23	645
1	22	23	649
2	22	23	638
3	22	23	642
4	22	23	648
5	22	23	643
6	22	23	649
7	22	23	599
8	22	22	603
9	22	22	600
10	21	22	609
11	21	22	535
12	22	23	601
13	22	22	627
14	21	23	532

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item, to the earthworm Eisenia foetida, in an artificial soil test has been investigated and gave a 14-Day LC50 value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the read across substance ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ to the earthworm (Eisenia foetida) in an artificial soil. The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 207 "Earthworm, Acute Toxicity Tests" and Commission Regulation (EC) No. 440/2008.

Methods.

Following a preliminary range-finding test, 60 earthworms (six replicates of 10 worms) were exposed to a single concentration of 1000 mg/kg of soil for a period of 14 days at a temperature of 21ºC to 23ºC. The number of mortalities were determined after 7 and 14 days exposure. A positive control using chloroacetamide, conducted approximately every 6 months, was reported for reference purposes.

Results.

The 14-Day LC₅₀ for the test item to earthworms (Eisenia foetida) based on nominal test concentrations was greater than 1000 mg/kg. The No Observed Effect Concentration was 1000 mg/kg.

These results are based on the soil dry weight.

The result of the positive control gave a 14-Day LC₅₀ for chloroacetamide of 43 mg/kg with 95% confidence limits of 41 - 45 mg/kg. The No Observed Effect Concentration was 18 mg/kg.

Exposure of the earthworm (Eisenia foetida) to the reference item gave 14-Day LC₅₀ values within the accepted range.

Conclusion.

The acute toxicity of the test item, to the earthworm Eisenia foetida, in an artificial soil test has been investigated and gave a 14-Day LC₅₀value of greater than 1000 mg/kg. Correspondingly the No Observed Effect Concentration was 1000 mg/kg.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Duration:

8 wk

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: survival, growth and reproduction rates

Remarks on result:

other: Survival, growth & reproduction of the adult’s the highest concentration without toxic effects (NOEC) was at least 1000 mg/kg ds. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg ds.

Details on results:

Survival of Adults and Adverse Effects
In the control group, the mortality rate of the adult test organisms after 28 days was 0% (Table1). Also at all test item concentrations up to and including 1000 mg/kg dry soil, the mortality rate was 0%.
Thus, the survival rate of the earthworm Eisenia fetida after 28 days of exposure was not affected up to and including the highest concentration of 1000 mg test item/kg dry soil. Moreover, no adverse effects were observed in the adult test organisms at any test concentration.

Growth of Adults
The mean body wet weights of the worms were determined per replicate at test start and of the surviving worms after the exposure period of 28 days are listed in Table 2. The individual wet weights of all test organisms at test start are given in the attached Appendix I.
The mean body weight of the adult worms in the control decreased during the 4-week exposure period on average by 8 mg per worm (-2% of the mean initial weight). At all test concentrations, the mean increase in body weight (0 to 4%) was statistically not significantly different than in the control (results of a Dunnett t-test, one-sided smaller, a = 0.05). The results for the growth of the test organisms correspond to the observation that no prominent difference in food consumption was determined between the control and all treatments up to and including the highest test concentration of 1000 mg test item/kg dry soil (Table 3).

Reproduction
The total number of living offspring, produced by all adults within 28 days of continuous exposure, is shown in Table 4 (expressed per test vessel and per surviving adult).

In the control, an average of 112 juvenile worms per test vessel were found (corresponding to a mean reproduction rate of 11.2 juveniles per surviving adult). The coefficient of variance of the reproduction rate per test vessel and per surviving individual in the control was 12%. Thus, the validity criteria of the test guidelines (at least 30 juveniles per test vessel, coefficient of variance of reproduction £30%) were well fulfilled.

At all test item concentrations up to and including 1000 mg/kg dry soil, the mean reproduction rates per surviving adult worm corresponded to 87-105% of the control value without a concentration-effect relationship. According to the results of a Dunnett t-test (one-sided smaller, a = 0.05), the mean reproduction rates were not statistically significantly reduced up to and including the highest test item concentration of 1000 mg/kg dry soil.
NOEC and LOEC
Taking into account the survival, consumption rates, growth and reproduction rates of the exposed adult test organisms, the highest concentration of the test item without toxic effects (NOEC) on Eisenia fetida after the test period was determined to be at least 1000 mg/kg dry soil. The NOEC might even be higher, but concentrations in excess of 1000 mg/kg dry soil were not tested. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.

Soil Moisture, pH, and Temperature
The soil moisture content was sufficiently constant (39-40% at test start and 46-47% at test termination after 8 weeks, Table 5). The pH value in the untreated artificial soil was 6.2 at the start of the test. At the test end, the pH in the test replicates was between 6.2 and 6.4. (Table 5) During the entire test period, the room temperature was in the range of 20 to 22 °C.

Results with reference substance (positive control):

Reference Item Derosal
At the single concentration of the reference item Derosal (8.33 mg formulation /kg dry soil = 5 mg Carbendazim/kg dry soil), the mortality rate of the adult worms was 2.5% (Table 1).
The mean decrease of 6% in body wet weight of the earthworms in the positive control during the exposure period was not statistically significantly different to the control (results of a Student-t-test, one-sided smaller, a = 0.05, Table 2).
The reproduction rate of Eisenia fetida in the positive control was almost completely inhibited and statistically significantly reduced in comparison to the control (Welch t-test, one-sided smaller, a = 0.05, Table 4).

Therefore, the results of the positive control demonstrated satisfactory test conditions.

TABLES

Table 1 Number of Living Adult Earthworms and % Mortality after 4 Weeks

Nominal test item concentration (mg/kg dry soil)	Vessel No.	Number of earthworms exposed	Number of living worms after 4 weeks	Sum of dead earthworms	% mortality
	1	10	10
	2	10	10
	3	10	10
Control	4	10	10
	5	10	10
	6	10	10
	7	10	10
	8	10	10	0	0
	1	10	10
10	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
32	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
100	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
320	2	10	10
	3	10	10
	4	10	10	0	0
	1	10	10
1000	2	10	10
	3	10	10
	4	10	10	0	0
Derosal 8.33 mg/kg dry soil	1	10	10
	2	10	9
	3	10	10
	4	10	10	1	2.5

 Table 2 Mean Body Wet Weights of Adult Earthworms at the Test Start and after 4 Weeks of Exposure

Nominal test item concentration	Vessel	Test start (Day 0)				After 4 weeks (Day 28)			Difference to test start
		Weight of all worms		No. of worms	Mean body weight/worm	Weight of all worms	No. of worms	Mean body weight/worm	Mean difference per worm
(mg/kg dry soil)	No.	(mg)			(mg)	(mg)		(mg)	(mg)	%	stat.^#
	1	4410		10	441	3984	10	398	-43	-10%
	2	4483		10	448	4422	10	442	-6	-1%
	3	4158		10	416	4130	10	413	-3	-1%
Control	4	4158		10	416	4162	10	416	0	0%
	5	4482		10	448	4174	10	417	-31	-7%
	6	4101		10	410	4080	10	408	-2	-1%
	7	4201		10	420	4372	10	437	17	4%
	8	3882		10	388	3939	10	394	6	1%
	Mean:				423			416	-8	-2%	¾
	SD:				21			17	20
	1	4492		10	449	4288	10	429	-20	-5%
10	2	4534		10	453	4480	10	448	-5	-1%
	3	3765		10	377	4067	10	407	30	8%
	4	4214		10	421	4368	10	437	15	4%
	Mean:				425			430	5	1%	n.s.
	SD:				35			17	22
	1	4691		10	469	4616	10	462	-8	-2%
32	2	4169		10	417	4235	10	424	7	2%
	3	4349		10	435	4587	10	459	24	5%
	4	3989		10	399	4420	10	442	43	11%
	Mean:				430			446	17	4%	n.s.
	SD:				30			18	22
	1	3871		10	387	3678	10	368	-19	-5%
100	2	4118		10	412	3945	10	395	-17	-4%
	3	3632		10	363	3730	10	373	10	3%
	4	4014		10	401	4310	10	431	30	7%
	Mean:				391			392	1	0%	n.s.
	SD:				21			29	23
	1	4377		10	438	4516	10	452	14	3%
320	2	4567		10	457	4496	10	450	-7	-2%
	3	4042		10	404	4242	10	424	20	5%
	4	4423		10	442	4647	10	465	22	5%
	Mean				435			448	12	3%	n.s.
	SD:				22			17	13
	1	4829		10	483	4602	10	460	-23	-5%
1000	2	4589		10	459	4577	10	458	-1	0%
	3	4098		10	410	4242	10	424	14	4%
	4	4052		10	405	4135	10	414	8	2%
	Mean:				439			439	0	0%	n.s.
	SD:				38			24	16
Derosal 8.33 mg/kg dry soil	1	4570		10	457	4372	10	437	-20	-4%
	2	4320		10	432	3413	9	379	-53	-12%
	3	3763		10	376	3657	10	366	-11	-3%
	4	3685		10	369	3509	10	351	-18	-5%
	Mean:				408			383	-25	-6%	n.s.^a
	SD:				43			38	19

Note: Calculations were performed with a commercial computer program (EXCEL). The results given in the table are rounded values.

^#Statistical comparison of the changes in mean body wet weight of the treatments with the control (absolute mean differences from test start to test end): results of a Dunnett t-test (one-sided,a= 0.05).

n.s.: Mean value not statistically significantly different compared to the control.

n.s.^a: Mean value not statistically significantly smaller than the control (results of a Student t-test, one-sided smaller,a= 0.05).

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of the substance without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be at least 1000 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.
The reproduction rate of Eisenia fetida in the positive control was almost completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions.

Executive summary:

The toxic effects of the read across substance ‘Distillates (Fischer-Tropsch), heavy, C18-50 - branched, cyclic and linear’ on survival, growth, and reproduction of the earthworm Eisenia fetida were assessed during a test period of eight weeks based on the OECD Guidelines for Testing Chemicals, Test No.222 (April 2004), the International Standards ISO 11268-2:1998(E) and the BBA Guideline “Richtlinien für die Prüfung von Pflanzenschutzmitteln im Zulassungsverfahren“ Part VI, 2-2, January 1994.

The test material was homogeneously mixed into artificial soil at the following test concentrations: 10, 32, 100, 320, and 1000 mg per kg dry soil. Additionally, a control and a reference item treatment were tested in parallel.

Adult earthworms were exposed in treated soils for a period of four weeks. After this period, the adults were removed from the test vessels and the survival and growth rate were determined. The cocoons and juvenile earthworms remained in the vessels for additional four weeks. The reproduction rate was determined by counting the number of offspring hatched from the cocoons after this additional test period of four weeks.

Summary of effects on earthworms exposed to the test substance:

Parameters	Control	test substance (mg/kg dry soil)					Derosal^® (mg/kg dry soil)
		10	32	100	320	1000	8.33
Mortality (%) after a 4-week exposure	0	0	0	0	0	0	2.5
Mean change in body fresh weight(%) after a 4-week exposure	-2	1	4	0	3	0	-6
Mean reproduction rate (juveniles per vessel)	112	117	112	97	114	106	0.75^a
Reproduction rate (% of control)	---	105	101	87	102	95	0.34

a: Statistically significant different from the control (Welch t-test, one-sided smaller, a=0.05).

In conclusion, taking into account the survival, growth and reproduction rates of the exposed adult test organisms, the highest concentration of the substance without toxic effects on Eisenia fetida (NOEC) after the test period was determined to be at least 1000 mg/kg dry soil. The lowest concentration with toxic effects (LOEC) could not be quantified, but was higher than 1000 mg/kg dry soil.

The reproduction rate of Eisenia fetida in the positive control was almost completely inhibited. Thus, the results of the positive control demonstrated satisfactory test conditions.

Description of key information

OECD Test Guideline 207(Acute Toxicity Test with Eisenia foetida): 
- LC50 (14-d): >1000 mg/kg dw test mat. (nominal) based on: mortality
- NOEC (14-d): ≥1000 mg/kg dw test mat. (nominal) based on: mortality
OECD Test Guideline 222 (Chronic Toxicity Test with Eisenia foetida): 
- LC50 (4-w): >1000 mg/kg dw test mat. (nominal) based on: growth, reproduction and mortality
- NOEC (4-w): ≥1000 mg/kg dw test mat. (nominal) based on: growth, reproduction and mortality

Additional information

A short-term earthworm toxicity study is available for the read across substance GTL Base Oil Distillates. The test was conducted in accordance with OECD 207 and GLP. Earthworms (Eisenia foetida) were exposed to a single concentration of the substance at a nominal loading rate of 1000 mg/kg of soil (dry weight) for a period of 14 days in artificial soil. There were no significant mortalities or sub-lethal effects during the 14-day exposure period. The 14-d acute LC₅₀ for earthworms was therefore determined to be greater than 1000 mg/kg dw.

A long-term toxicity study with E. foetida has been conducted with the read across substance GTL Base Oil Distillates in accordance with GLP and OECD 222. Adult test organisms were exposed to 10, 32, 100, 320 and 1000 mg/kg dw soil for 4 weeks and then removed, while the juvenile earthworms remained in the vessels for an additional 4 weeks. Adult mortality and mean change in body weight were recorded, as well as mean reproduction rate of juveniles. Based on these parameters an LC₅₀ and a LOEC could not be determined and a NOEC of at least 1000 mg/kg dw has been determined.