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Toxicity to soil macroorganisms except arthropods

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Endpoint:
toxicity to soil macroorganisms except arthropods: short-term
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Oct. 4, 1989-Oct. 18, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study done according to OECD Guideline 207.
Qualifier:
according to
Guideline:
OECD Guideline 207 (Earthworm, Acute Toxicity Tests)
GLP compliance:
yes (incl. certificate)
Details on sampling:
Samples were from the 250 mg/kg dw concentration taken on day 0, 7, and 14. Measured concentrations were 235, 211, and 40.5 mg/kg dw respectively.
Vehicle:
no
Details on preparation and application of test substrate:
Test material was mixed with 1.16 L of deionized water, and then added to 2.44 kg of artificial soil.
Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:
TEST ORGANISM
- Common name: Earthworm
- Source: Vittor and Associates, Mobile Alabama
- Age at test initiation (mean and range, SD): adult
- Weight at test initiation (mean and range, SD): average 0.66 g

Study type:
laboratory study
Substrate type:
artificial soil
Total exposure duration:
14 d
Test temperature:
19-22 degrees C
pH:
5.5-5.8
Moisture:
34-36%
Details on test conditions:
TEST SYSTEM
- Test container (material, size): jars with perforated lids
- Amount of soil or substrate: 900 g moist soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 4


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil texture (if natural soil)
- % sand: 70
- % clay: 20, also 10% sphagnum peat
- Soil classification system: sandy loam


OTHER TEST CONDITIONS
- Photoperiod: constant lighting
- Light intensity: ~600 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality, health, behaviour, and number of cocoons on days 7 and 14

Nominal and measured concentrations:
Nominal: 0, 63, 125, 250, 500, and 1000 mg/kg dw
Duration:
14 d
Dose descriptor:
NOEC
Effect conc.:
250 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: body weight
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
Duration:
14 d
Dose descriptor:
EC10
Effect conc.:
277 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: body weight
Remarks on result:
other: EC10 was not reported in original study report, but later calculated using study results using the method of Vanewijk, PH, and Hoekstra, JA, 1993. Calculation of the EC50 and its confidence interval when subtoxic stimulus is present. Ecotoxicol. Environ
Details on results:
There was 5% mortality at the 1000 mg/kg dw concentration on day 14. Body weight was decreased in the 500 and 1000 mg/kg dw groups by 23 and 33% respectively. The 1000 mg/kg dw group also showed lethargy and burrowing was also affected. Cocoon production was diminished in the 250 mg/kg dw group, and inhibited at higher concentrations.

Mortality and Weight Loss of Earthworms

Concentration

Number of

% Weight Loss

(mg/kg dw)

Day 0

Day 7

Day 14

Day 0-14

Control

40

40

40

14

63

40

40

40

5

125

40

40

40

6

250

40

40

40

5

500

40

40

40

23

1000

40

38

37

33

Conclusions:
The 14-day LC50 to earthworms was > 1000 mg/kg dw soil. The 14-day NOEC was 250 mg/kg dw soil.
Executive summary:

Groups of earthworms were exposed to concentrations of 0, 63, 125, 250, 500, and 1000 mg/kg dw for 14 days. The test animals were examined for mortality, behaviour, health, and reproduction on days 7 and 14. 5% mortality was seen at the highest concentration. Effects to health were seen at the 500 and 1000 mg/kg dw concentrations. The 14 -day LC50 was > 1000 mg/kg dw, and the NOEC was 250 mg/kg dw.

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented journal article.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Draft ISO/WD 16387 protocol
Qualifier:
according to
Guideline:
ISO 11268-2 (Effects of Pollutants on Earthworms. 2. Determination of Effects on Reproduction)
Deviations:
yes
Remarks:
limited cocoons Comparable to ISO 11268-2, but with only weight measurement
Principles of method if other than guideline:
Adult E. albidus (10) were placed in soil containing the test substance for 21 days. The adults were removed, and the cocoons incubated in the soil for another 21 days. The juveniles were then counted.
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
not specified
Details on preparation and application of test substrate:
Earthworm tests: For the reproduction tests, 1 kg dry weight of soil was carefully mixed with 160 mL of LAS solution using an electric mixer and filled into plastic pots. For the growth test with juvenile A. caliginosa, 60 g dry weight of soil were mixed with 9.6 mL of LAS solution with a spatula and filled into 160-mL polyethylene beakers with perforated lids for ventilation.
Enchytraeid test: Forty grams dry weight of soil were mixed with 6.4 mL of LAS solution and filled into 160-mL beakers with perforated lids for ventilation.
Test organisms (species):
other: Enchytraeus albidus, Aporrectodea caliginosa, Aporrectodea longa
Animal group:
annelids
Details on test organisms:
For the growth test with juvenile A. caliginosa (2-3 weeks old),
Study type:
other: no data
Substrate type:
other: Sandy, agricultural soil
Limit test:
no
Post exposure observation period:
21 days (28 days for A. caliginosa and A. longa growth tests)
Details on test conditions:
Earthworm tests: The six treatments consisted of one control and five concentrations of LAS and these treatments were randomly assigned to the experimental units. After 24-hour equilibration of the test soil, 3 (rather than 10) earthworms were added to closed containers with perforated lids for ventilation. Approximately 5 g per worm were added after the test animals had been introduced. The containers were then incubated for 21 days in darkness and the contents were later wet sieved through a 1-mm mesh. Water content was adjusted after 14 days. For the growth test with juvenile, six treatments consisted of one control and five concentrations of LAS and these treatments were randomly assigned to the experimental units. After 24-hour equilibration of the test soil, one earthworm was added to each container. The beakers were incubated for 28 days in darkness and then the earthworms were recovered and their guts were cleared. The surviving animals were dried for 24 hours and their dry weight was recorded to the nearest 0.1 mg. The examination of the effects on growth of A. longa used the same method except the test period was 42 days.
Enchytraeid: After 24-hour equilibration of the test soil, 10 adult E. albidus were added to each container and incubated in darkness for 21 days. After incubation, the surviving adult animals were removed from the soil. Now only containing cocoons, the soil was incubated in the beakers for another 21 days to allow development and hatching of the juveniles. After this period, the soil containing juveniles was stained with Bengal red, and water was added to facilitate counting of the juveniles.
Nominal and measured concentrations:
Enchytraeid: The test concentrations were not provided but can be estimated from Figure 5 to be 0, 20, 40, 80, 200 and 400 mg/kg with the numbers of adults surviving per replicate to be approximately 10, 10, 10, 10, 9, and 6, and the numbers of juveniles per replicate (reproduction) to be approximately 77, 50, 37, 21, 0, and 0, respectively.
Reference substance (positive control):
not specified
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
6.2 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: E. albidus
Details on results:
It should be noted that these studies report worst case exposures due to the use of a sandy test soil and the fact that LAS was added as an aqueous solution to the soil. In addition, too few replicates were used for the ECx approach (e.g., <5 controls) and several key deviations from draft protocols limited the reliability of endpoints for some studies (e.g., A. caliginosa and A. longa cocoon production). Nominal concentrations were derived from tables and figures since actual values were not found in the text.

The following table shows the results of all tests.  All values are nominal LAS concentrations in mg/kg dry weight.

Species

Parameter

NOEC

LOEC

LC10 or EC10

LC50 or EC50

Relia-bility Rating

Rationale for Reliability Rating

Enchytraeus albidus

Survival, adults

Reproduction

198

20

397

40

194

6

430

41

1

1

Draft ISO/WD 16387 protocol

Aporrectodea caliginosa

Survival, adults

Cocoon production

Survival, juveniles

Growth, juveniles

278

>793

>397

278

793

>793

>397

397

329

14

>397

105

535

129

>397

354

2

3

2

2

Comparable to

ISO 11268-2

Deviations; limited cocoons

Comparable to ISO 11268-2, but with only weight measurement

Aporrectodea longa

Survival, adults

Cocoon production

Survival, juveniles

Growth, juveniles

278

>793

397

79

793

>793

793

278

329

27

296

84

535

137

517

349

2

3

2

2

Comparable to

 ISO 11268-2

Deviations; limited cocoons

Comparable to ISO 11268-2, but with only weight measurement

Folsomia fimetaria

Survival, adults

Reproduction

>793

278

>793

278

>793

85

>793

424

1

1

Comparable to  ISO 11267

Hypogastrura assimilis

Reproduction

79

278

99

421

1

Comparable to  ISO 11267

Hypoaspis aculeifer

Survival, adults

Reproduction

>793

278

>793

793

>793

82

>793

236

2

2

No guideline available

Conclusions:
The 21-day EC10 to E. albidus was 6.2 mg/kg dry weight.
Executive summary:

Adult E. albidus (10) were placed in soil containing the test substance for 21 days. The adults were removed, and the cocoons incubated in the soil for another 21 days. The juveniles were then counted. The 21-day EC10 to E. albidus was 6.2 mg/kg dry weight.

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented, peer-reviewed publication.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Similar to ISO (1998) and ISO 16387 (2004).
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
Initial concentrations were measured.
Vehicle:
yes
Details on preparation and application of test substrate:
LAS was mixed in demineralized water, and then added to dry Askov soil. Fresh sludge (approx. 200 g) was freeze-dried. This was crushed and sieved through a 2 mm sieve. A methanol solution of LAS was then added to the sludge.
Test organisms (species):
other: A. caliginosa, E. crypticus
Details on test organisms:
TEST ORGANISM
- Common name: earthworm
- Source: surrounding of National Environmental Research Institute, Silkeborg, Denmark

ACCLIMATION
- Acclimation period: 7 days

TEST ORGANISM
- Source: permanent laboratory cultures

Study type:
laboratory study
Substrate type:
natural soil
Total exposure duration:
28 d
Test temperature:
16 degrees C
Details on test conditions:
A. caliginosa
TEST SYSTEM
- Test container (material, size): plastic pots
- Amount of soil or substrate: 1 kg soil and 80 ml test solution
- No. of organisms per container (treatment): 4
- No. of replicates per treatment group: 6
- No. of replicates per control: 2

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Askov
- Soil texture (if natural soil)
- % sand: 76
- % silt: 12
- % clay: 11
- Soil classification system: sandy loam
- Organic carbon (%):1.3
- CEC: 13.4


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cocoons were collected by sieving with 1 mm mesh after 28 days.



E. crypticus

- Amount of soil or substrate: 20 g soil and 3 ml test solution
- No. of organisms per container (treatment): 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Askov
- Soil texture (if natural soil)
- % sand: 76
- % silt: 12
- % clay: 11
- Soil classification system: sandy loam
- Organic carbon (%):1.3
- CEC: 13.4
Nominal and measured concentrations:
A. caliginosa: 25-150 mg/kg soil (nominal)
E. crypticus 0, 10, 40, 60, 80, 100, 120 mg/kg soil (nominal)
0.65, 7.1, 26.3, 45.5, 60.3, 92.4, 102.7 mg/kg soil (measured)
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
46 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
reproduction
Remarks on result:
other: A. caliginosa
Dose descriptor:
EC10
Effect conc.:
>= 120 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
reproduction
Remarks on result:
other: E. crypticus
Details on results:
No toxicity to E. crypticus was seen at any dose level, including the highest dose level of 120 mg/kg soil. A dose-related inhibition of cocoon production was seen in A. caliginosa at the highest concentration of 150 mg/kg soil.
Conclusions:
The EC10 for A. caliginosa was 46 mg/kg soil. The EC10 for E. crypticus was >=120 mg/kg soil.
Executive summary:

Groups of A. caliginosa were exposed to soil containing 25 -150 mg/kg soil of LAS for 28 days. Groups of E. crypticus were exposed to concentrations of 0, 10, 40, 60, 80, 100, or 120 mg/kg soil (nominal). EC10 values based on reproduction were 46 mg/kg soil for A. caliginosa, and >= 120 mg/kg soil for E. crypticus.

Description of key information

A series of studies have been conducted on soil macroorganisms that include or exclude arthropods. The primary route of entry for LAS into the terrestrial environment is land application of sewage sludge intended as an amendment to agricultural lands. A large number of terrestrial ecotoxicology studies were reviewed by Jensen et al. (2007) to develop a European wide risk assessment of LAS in agricultural soils. Nine invertebrate species, three oligochaetes and six arthropods, have been evaluated in chronic toxicity to LAS in soil. Endpoints were all considered sensitive for these taxa. Only growth and reproduction endpoints were used in the PNEC derivation for soil. Effects on oligochaetes (Table 37) and arthropods (Table 38) were highly overlapping. NOECs or EC10s for oligochaetes varied from 27-250 mg/kg soil. Key studies on the oligochaete Aporroectodea caliginosa and the springtail Folsomia fimeteria were conducted by Holmstrup and Krogh (1996) and Holmstrup et al. (2001) in several soil types including sand, loam and clay with varying dominant cations. A. calignosa had EC10s that ranged from 44-105 with a geometric mean of 71.7 mg/kg soil for the growth endpoint (most sensitive) and F. fimeteria had EC10s that ranged from 85-161 mg/kg soil for the reproduction endpoint (most sensitive). Studies on Enchytraeidae (Enchtraeus sp.) were conducted by Holmstrup and Krogh 2001 and Krogh et al. 2007 to determine the effects of pollutants on reproduction and survival, with an EC10 of 27 mg/kg soil, the geometric mean of 2 EC10 values.

Key value for chemical safety assessment

Additional information