Amines, polyethylenepoly-

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:

1-1-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study is complete and well executed

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

A stock solution was prepared by weighing 4680 mg of REPA
S140 using an analytical balance and deionised water was
added to reach a fmal net weight of 800 g. The resulting
suspension contained a concentration of 5.85 mg test item/g. A
magnetic stirrer was used to obtain a homogenous dispersion of
the test item.
The following amounts of this stock solution were added to
2050 g dry artificial soil to prepare the target nominal
concentrations of the test item in the artificial soil

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

age: 11 to 12 months, with well developed clitellum, age range
between test individuals not differing by more than 4 weeks
BW: 300 mg to 579 mg
Origen: Bred under standardised conditions (see 207, in a
breeding medium of cattle manure, peat, sand, calcium
carbonate and straw, fed with cattle manure, stored at room
temperature) by IBACON
Acclimatisation: I day, in artificial soil, under test conditions

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

Within the range of 18°C to 22 °C

pH:

pH was checked at the start and end of the main test for each
test concentration (CaCh, ISO 10390);
pH range at experimental start: 5.6 to 5.8 and thus did not
deviate from the values given by the guidelines (6.0 ± 0.5)
pH at experimental end: 6.0

Moisture:

Once a week the water content of the soil was checked by
weighing each container and evaporated water was
replenished, if necessary. In this way, it was ensured that the
difference in water content between experimental start and
end was less than 10%.

Details on test conditions:

number of replicates: 8 per control, 4 per test item treated group
number of individuals: 10 per replicate, i.e. 80 individuals per control, 40 individuals
per test item treated group
introduction of individuals: All worms were washed with tap water, dried with a dry paper
towel, weighed individually and randomly assigned to batches
of 10 worms. The different batches were sorted into four
classes on the basis of the total weight and one batch of each
weight class was assigned to each treatment group (two batches
for the control) to ensure weights were homogeneous. The
earthworms were placed on the surface of the artificial soil
after application.
test duration: 8 weeks
exposure time of adults: After 4 weeks, the artificial soil was transferred to a tray and
adult worms were counted, removed and weighed per replicate
after they had been washed under tap water and dried on a dry
paper towel. Missing earthwonns and earthworms that failed to
respond to gentle stimulation were considered to be dead. The
remaining soil (without the adult worms) was then returned to
the respective test containers.
exposure time offspring: Additional 28 days
removal of juvenals: Juveniles were removed by placing the test units in a water bath
at 50 - 60°C and counting all emerging worms. In addition the
soil of each container was emptied out onto a tray and checked
visually for any remaining young worms.

Nominal and measured concentrations:

100, 180, 320, 560, 1000 mg/kg

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mortality

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Details on results:

Control mortality was 0% and so this validity criterion was met.
The number of juvenile worms per replicate was 281 to 440 and so this validity criterion was met.
Was 13.6% and so this validity criterion was met.

Results with reference substance (positive control):

In the most recent test with the reference item Luxan Carbendazim 500 FC (perfonned under IBACON
Study Number 46642022 from December 2009 to February 2010), there were statistically significant
effects on reproduction at a concentration of 1.5 mg carbendazim/kg soil and higher; the ECso for
reproduction was calculated as 1.47 mg carbendazim/kg soil. The results are shown in Appendix 2
indicating that the sensitivity of the wonns was consistent with the level proposed by the OECD 222
guideline (significant effects between 1 and 5 mg carbendazim/kg soil).

Reported statistics and error estimates:

(Dunnett's t-test, a = 0.05, one-sided smaller)

None of the body weight changes in the test item treated groups were significantly different

compared to the control, except at the concentration of 180 mg/kg soil the body weight was

significantly reduced compared to the control. The statistical significance at the concentration of

180 mg test item/kg soil was not considered to be treatment related since at the higher concentrations

no significant differences in body weight could be observed (Dunnett's t-test, a = 0.05, two-sided). The

NOEC for body weight changes was determined to be 1000 mg test item/kg soil.

A mean of 374 juvenile worms was produced in the control. In the containers treated with HEPA S140,

the mean reproduction was 241 to 322 juvenile worms. The reproduction rates at the test concentrations

of 100 and 320 mg test item/kg soil were not significantly reduced compared to the control. At the test

concentrations of 180, 560 and 1000 mg/kg soil the reproduction compared to the control was

statistically significantly different (Dunnett's t-test, a = 0.05, one-sided smaller). However, the

statistical significance at the concentrations of 180, 560 and 1000 mg test item/kg soil was not

considered to be treatment related since no dose relation was observed.

In all treatment groups, food was consumed. The results show

that the turnover of biomass of those earthworms exposed to

the different rates of the test item was comparable to the

control, except at 1000 mg test item/kg soil the food intake was

slightly reduced.

No behavioural abnormalities were observed and all worms

burrowed into the soil within 15 minutes after introduction.

Luxan Carbendazim 500 FC 1.6 2.3 3.5 5.3 7.9[mg/kg soil] Control [mg a.i.lkg soil] 0.69 1.0 1.5 2.3 3.4 Mortality (day 28) [%] 0.0 0.0 0.0 0.0 0.0 2.5 Significance 1) --n.s. n.s. n.s. n.s. n.s. Weight change (day 28) [%] 35.0 31.3 38.1 27.2 12.4 -11.8 Significance 2) --n.s. n.s. n.s. * * No. ofjuveniles (day 56) 213 204 213 95 21 0 Significance2 ) --n.s. n.s. * * * Reproduction in [%] of 95.8 100 44.6 9.8 0control (day 56) --Food consumption [g] 24.4 24.3 24.5 23.0 21.3 16.3 Endpoints [mg a.i.lkg soil] NOEC (day 28 mortality) 3.4 NOEC (day 28 weight) 1.5 NOEC (day 56 reproduction) 1.0 ECso for reproduction 1.47 95% confidence limits 1.20 to 1.76

�

Validity criteria fulfilled:

yes

Conclusions:

study is complete and well performed, all criteria aremet and the NOEC is 1000 mg/kg

Executive summary:

The toxicity of HEPA to earthworms was assessed according to the OECD 222 guideline. The body weight change of the earthworms after 4 weeks exposure to HEPA S140 was not statistically

significantly different compared to the control up to and including the test concentration of 1000 mg/kg

soil, except at the concentration of 180 mg/kg soil the body weight was significantly reduced compared

to the control (Dunnett's t-test, a = 0.05, two-sided). The statistical significance at the concentration of

180 mg test item/kg soil was not considered to be treatment related since at the higher concentrations no

significant differences in body weight compared to the control were observed.

The reproduction rates were statistically significantly different compared to the control at the

concentrations of 180, 560 and 1000 mg/kg soil (Dunnett's t-test, a = 0.05, one-sided smaller). This

statistical significance was not considered to be treatment related since no dose relation was observed.

Therefore the overall NOEC was proposed to be 1000 mg test item/kg soil. No behavioural abnormalities were observed in any of the treatment groups. The feeding activity in all

the treated groups was comparable to the control, except at 1000 mg test item/kg soil the food intake was

slightly reduced.

In an earthwonn reproduction and growth study with HEPA S140 the No Observed Effect Concentration

(NOEC) for mortality and growth of the earthwonn Eisenia fetida was determined to be 1000 mg test

item/kg soil, i, e. the highest concentration tested.

The No Observed Effect Concentration (NOEC) for reproduction was proposed to be the concentration

of 1000 mg test item/kg soil.

Description of key information

OECD Guideline 222 Eisenia fetida: NOEC (56 d): 1000 mg/kg soil dw based on reproduction.

Additional information

The toxicity of PEPA (CAS 68131-73-7) to earthworms was tested according to OECD guideline 222.

The test substance was added to artificial soil and adult earthworms of the species Eisenia fetida were placed on the soil surface. The tested concentrations were 100, 180, 320, 560 and 1000 mg test item/kg soil dry weight. The total test period was 8 weeks. After 28 days the adult earthworms were removed from the treatments. The number of earthworms as well as symptoms of intoxication was recorded. After an additional exposure period of 28 days the reproduction rate (number of juveniles) was determined. Under the conditions of this study the substance did not induce significant mortality, pathological symptoms or changes of the behaviour of adult earthworms after 28 days of exposure at all tested concentrations.

The reproduction rate was statistically significantly lower at the test item concentrations 180, 560 and 1000 mg test item/kg soil dry weight than in the untreated control. A dose relation was not observed and thus the observed decrease was not related to treatment effects. Thus, a No Observed Effect Concentration (NOEC) of 1000 mg test item/kg soil dry weight based on the reproduction rate was determined.