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Ecotoxicological information

Toxicity to soil macroorganisms except arthropods

Administrative data

Endpoint:
toxicity to soil macroorganisms except arthropods: long-term
Remarks:
Effects on Reproduction and Growth of the Earthworm
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 2016 to January 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
-
EC Number:
446-990-1
EC Name:
-
Molecular formula:
C5H11O5 [C6H10O5]1.45 and C5H10O4 and C5H12O5
IUPAC Name:
2-(hydroxymethyl)oxolane-3,4-diol; 5-{[2,3,4,5-tetrahydroxy-6-(xenoniooxy)hexyl]oxy}pentane-1,2,3,4-tetrol; pentane-1,2,3,4,5-pentol
Specific details on test material used for the study:
Purity/Concentration: >99% dry matter
Batch number: 160722014882 w/o solvent
Appearance: Beige waxy liquid
Storage conditions: Room temperature (15 - 25°C)

Sampling and analysis

Analytical monitoring:
no
Details on sampling:
not applicable

Test substrate

Vehicle:
no
Details on preparation and application of test substrate:
Range finding test:
Prior to treatment the initial moisture content of the soil was determined as 27.44% and the volume required to achieve 35% moisture content calculated as 83.16 mL water/1100 g dry soil. The test substance was added to the soil in a volume of 83.16 mL, and mixed in using a hand held electric mixer.

Difinitive test:
Prior to treatment the initial moisture content of the soil was determined as 27.44% and the maximum water holding capacity (MWHC) as 76.74%. Moisture content equivalent to 55% of MWHC was selected as providing suitable conditions for earthworm development. No pre-moistening of the soil was required. The volume required to achieve 55% of the maximum water holding capacity was calculated as 325 mL water/2200 g dry ISO earthworm soil.
The amount of test item required to achieve the final soil concentration was added as a solution in 200 mL of reverse osmosis with an additional 125 mL of reverse osmosis water to bring the soil moisture content to 55% MWHC. The treated soil was then mixed in using a hand held electric mixer.

Test organisms

Test organisms (species):
Eisenia fetida
Animal group:
annelids
Details on test organisms:

TEST ORGANISM
- Common name: Earthworm
- Source: reputable supplier; details are recorded only in the raw data to maintain supplier confidentiality
- Age at test initiation (mean and range, SD): adult worms selected based on weight range 300- 600 mg


ACCLIMATION
- Acclimation period: 24 hrs in untreated artificial soil
- Acclimation conditions: temperature 17.8 – 21.9°C and lighting 410-795 lux
- Health during acclimation (any mortality observed): not stated in report

Study design

Study type:
laboratory study
Substrate type:
artificial soil
Limit test:
no
Total exposure duration:
56 d
Remarks:
Range finding test duration: 14 d. Definitive experiment duration: Adults: 28 d, Juveniles: 56 d
Post exposure observation period:
not applicable

Test conditions

Test temperature:
Range finding test:17.8-21.5°C
Definitive test: 18-5-21.9°C
pH:
6.5-6.7
Moisture:
At termination maximum water holding capacity (MWHC) group replicate mean moisture content values were found to be approximately 39.87 - 45.66%, equivalent to 51.98 - 59.53% MWHC.
Details on test conditions:
TEST SYSTEM
- Test container (material, size):
Range finding test: one-litre glass containers covered with perforated polythene
Definitive test: plastic containers measuring approximately 11 x 17 x 5 cm, each covered with a tight fitting lid.
- Amount of soil or substrate:
Range finding test: 1100g dry soil mixed with 83.16ml test substance was divided between two containers
Definitive test: 711.0 g moist soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: range finding test: 2, definitive test: 4
- No. of replicates per control: definitive test: 8
- No. of replicates per vehicle control: range finding test: 1, definitive test: 8

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Artifical soil: Batch WS1602
- Composition: Industrial quartz sand 70 % w/w, Kaoilin clay: 20% w/w, Peat 10% w/w

OTHER TEST CONDITIONS
- Photoperiod: Range finding test: continuous light (410-653 lux), defintive test:16hr light: 8hr dark (468-795 lux)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Range finding test - Health: Any earthworms visible on the soil surface were observed daily for behavioural and pathological signs.
- Mortality: On Days 7 and 14, the medium was emptied from the test containers and the earthworms removed for counting. On Day 7, the medium was returned to the containers and surviving earthworms replaced on the surface.
- Bodyweight: Bodyweights of earthworms were recorded in both treatment and control replicates prior to treatment and on Day 14.
Defintive test - Counts of worms and mortalitities: On Day 28 of the study the soil was removed from the containers. The numbers of live adults were recorded. The soil was returned to the containers and the adult worms were humanely euthanized by freezing before being discarded.
- Health: Any worms visible on the soil surface were observed daily for behavioural and pathological signs.
- Weights: Adult worms were weighed individually and then in replicates of ten worms prior to treatment and at the end of the four week adult treatment period in replicates of ten.
- Juvenile worms: The number of surviving juvenile worms in each replicate was determined on Day 56.



TEST CONCENTRATIONS
Range finding test: 0.1, 1, 10, 100 and 1000 mg/kg dry soil
Difinitive test: 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil
Nominal and measured concentrations:
Range finding test: 0.001323, 0.01323, 0.1323, 1.323 and 13.23 mg/ml (nominal)
Definitive test: 0.180, 0.324, 0.582, 1.048, 1.886, 3.395, 6.111 and 11.000 mg/ml (nominal)
Reference substance (positive control):
yes
Remarks:
Mascot Systemic (active ingredient: Carbendazim)

Results and discussion

Effect concentrationsopen allclose all
Duration:
28 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:
28 d
Dose descriptor:
LC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: bodyweight
Duration:
56 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks:
number of juveniles
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
56 d
Dose descriptor:
NOEC
Effect conc.:
1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
reproduction
Details on results:
Range Finder:
No mortality was observed in the water control or at any D-glucopyranose treatment rate in comparison to the water control.

Health and Mortality:
No mortality was observed in the water control or at any D-glucopyranose treatment rate in comparison to the water control.
A NOEC of 1000 mg/kg dry soil was achieved.

Bodyweights:
There was no statistically significant reduction in the mean bodyweights 28 days after treatment at any D-glucopyranose rate compared to the water control.
The adjusted mean bodyweights on Day 28 at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil were 517, 489, 499, 507, 509, 510, 551 and 522 mg respectively compared to 493 mg in the water control.
A NOEC of 1000 mg/kg dry soil was achieved.

Juvenile Worms
Control group productivity was acceptable (mean of 234 juveniles per replicate). The coefficient of variation of the number of juveniles in the water control group was 4.37%.
The mean number of juveniles produced at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil was 253, 232, 221, 242, 234, 227, 230 and 256 respectively compared to 234 in the water control.
There was no statistically significant reduction in the number of juveniles produced at any treatment rate compared against the water control.
A NOEC of 1000 mg/kg dry soil was achieved compared against the water control.
Results with reference substance (positive control):
Health and Mortality:
Mortality of 1.25% was recorded in the toxic reference treatment at 28 days.

Bodyweights:
The toxic reference group had a significantly (p<0.001***) lower body weight compared against the water control group.
The adjusted mean bodyweight on Day 28 was 345 mg in the toxic reference.

Juvenile Worms
The mean number of juveniles produced was 0.25 for the toxic reference.
There was a statistically significant reduction (p<0.001***) in the mean number of juveniles of the Mascot Systemic group at 5 mg a.i./kg dry soil.

Any other information on results incl. tables

% Adult Mortality and Treatment Mean Bodyweights (mg) are displayed in the table below

Treatment

(mg/kg dry soil)

% Mortality

Day of study

Change

pvalues

Time 0

Day 28(a)

(%)

Water control

0

0

344

493

+43.3

-

D-glucopyranose

16.3

0

343

517

+50.7

0.829

29.4

0

343

489

+42.6

0.829

52.9

0

345

499

+44.6

0.829

95.3

0

344

507

+47.4

0.652

171.5

0

345

509

+47.5

0.554

308.6

0

345

510

+47.8

0.534

555.6

0

344

551

+60.2

0.041

1000

0

343

522

+52.2

0.041

Mascot Systemic

5 mg a.i./kg dry soil

1.25

345

345

0

<0.001***

(a)Mean adjusted bodyweight

- not applicable

pvalues are for comparison with water control using Williams’ test for the treatment rates and t-test for the Mascot Systemic

***p< 0.001

Mean Number of Juvenile Worms per Treatment Replicate are displayed in the table below

Treatment

(mg/kg dry soil)

Day 56

pvalue

Water control

0

234

-

D-glucopyranose

16.3

253

0.177

29.4

232

0.999

52.9

221

0.409

95.3

242

0.999

171.5

234

>0.999

308.6

227

0.727

555.6

230

0.729

1000

256

0.729

Mascot Systemic

5 mg a.i./kg dry soil

0.25

<0.001***

- not applicable

All groups were compared against the water control group using Dunnett’s test

***p< 0.001

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The preliminary range finding study indicated that the LC50 value was >1000 mg/kg dry soil. No mortality was observed in the water control or at any treatment rate at seven or 14 days after treatment. There were slight reductions in bodyweight of 1.3 and 1% at 1 and 10 mg/kg dry soil respectively in comparison to the water control group. All worms appeared healthy.

In the reproduction study no mortality was observed in the water control or at any D-glucopyranose treatment rate. Mortality of 1.25% was recorded in the toxic reference treatment at 28 days.
The LC50 for adult mortality at Day 28 was >1000 mg/kg dry soil. The confidence intervals could not be obtained. A NOEC of 1000 mg/kg dry soil was achieved.

There was no statistically significant reduction in the mean bodyweights 28 days after treatment at any D-glucopyranose rate compared to the water control. A significantly lower body weight (p<0.001***) was recorded in the toxic reference group in comparison to the water control.

The adjusted mean bodyweights on Day 28 at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil were 517, 489, 499, 507, 509, 510, 551 and 522 mg respectively compared to 493 mg in the water control and 345 mg in the toxic reference.

The LC50 for mean adult bodyweight was >1000 mg/kg dry soil. The confidence intervals could not be obtained. A NOEC of 1000 mg/kg dry soil was achieved.

There was no statistically significant reduction in the number of juveniles produced at any treatment rate compared against the water control.

The mean number of juveniles produced at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil was 253, 232, 221, 242, 234, 227, 230 and 256 respectively compared to 234 in the water control and 0.25 for the toxic reference.

The EC50 for the number of juveniles on Day 56 was >1000 mg/kg dry soil. A NOEC of 1000 mg/kg dry soil was achieved compared against the water control.

The study was considered valid as there was ≤ 10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition application of the toxic reference Mascot Systemic at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.
Executive summary:

A preliminary range finding study was conducted at 0.1, 1, 10, 100 and 1000 mg/kg dry soil to determine the acute toxicity of D-glucopyranose, oligomers, xylityl glycosides and 1,4 anhydro D-xylitol and D-xylitolto the earthworm Eisenia fetida and to ensure selection of appropriate treatment rates for the definitive reproduction study.

A study was then performed to determine the effects of D-glucopyranose, oligomers, xylityl glycosides and 1,4 anhydro D-xylitol and D-xylitolon the reproduction and growth of the earthworm, Eisenia fetida, in an artificial soil under laboratory conditions. The method followed was that described in OECD 222 Guideline for the testing of Chemicals, Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei) 29 July 2016.

The test substance name D-glucopyranose, oligomers, xylityl glycosides and 1,4 anhydro D-xylitol and D-xylitol will be referred to as D-glucopyranose throughout the remaining report.

Ten groups of worms were allocated to the reproduction study. Eight groups of 40 worms were treated with D-glucopyranose at test rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil weight, mixed into the soil. A negative control group of 80 worms, was maintained in untreated soil, and a positive control group of 80 worms, was treated with Mascot Systemic (active ingredient carbendazim) at 5 mg a.i./kg dry soil weight. Adult worms were removed from the soil four weeks after treatment and the juvenile worms reared for a further four weeks.

Findings

The preliminary range finding study indicated that the LC50value was >1000 mg/kg dry soil. No mortality was observed in the water control or at any treatment rate at seven or 14 days after treatment. There were slight reductions in bodyweight of 1.3 and 1% at 1 and 10 mg/kg dry soil respectively in comparison to the water control group. All worms appeared healthy.

In the reproduction study no mortality was observed in the water control or at any D-glucopyranose treatment rate. Mortality of 1.25% was recorded in the toxic reference treatment at 28 days.

The LC50for adult mortality at Day 28 was >1000 mg/kg dry soil. The confidence intervals could not be obtained. A NOEC of 1000 mg/kg dry soil was achieved.

There was no statistically significant reduction in the mean bodyweights 28 days after treatment at any D-glucopyranose rate compared to the water control. A significantly lower body weight (p<0.001***) was recorded in the toxic reference group in comparison to the water control.

The adjusted mean bodyweights on Day 28 at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil were 517, 489, 499, 507, 509, 510, 551 and 522 mg respectively compared to 493 mg in the water control and 345 mg in the toxic reference.

The LC50for mean adult bodyweight was >1000 mg/kg dry soil. The confidence intervals could not be obtained. A NOEC of 1000 mg/kg dry soil was achieved.

There was no statistically significant reduction in the number of juveniles produced at any treatment rate compared against the water control.

The mean number of juveniles produced at rates of 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg/kg dry soil was 253, 232, 221, 242, 234, 227, 230 and 256 respectively compared to 234 in the water control and 0.25 for the toxic reference.

The EC50for the number of juveniles on Day 56 was >1000 mg/kg dry soil. A NOEC of 1000 mg/kg dry soil was achieved compared against the water control.

The study was considered valid as there was≤10% adult mortality at four weeks and ≥30 juveniles had been produced in each water control replicate by the end of the test with the coefficient of variation of reproduction ≤ 30%. In addition application of the toxic reference Mascot Systemic at 5 mg a.i./kg dry soil resulted in substantial and unequivocal toxic effects.