Polyethylene polyamine, pentaethylenehexamine fraction

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:

2014-08-28 to 2014-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

no critical deviations

Principles of method if other than guideline:

Food was provided on day 0 (test start) instead of day 1 due to good experience with this procedure. The soil moisture deviated by more than 10 % from the initial value in the control and all test item concentrations. On day 28 the earthworms were washed with dechlorinated tap water, dabbed dry on paper towels, counted and weighed.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil
The appropriate test item quantity was weighed out for each test item concentration and dissolved in demineralised water.
The spiking solutions and additional demineralised water were added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 54 % of the maximum water holding capacity.
Subsequently, the test medium was thoroughly mixed
to ensure a homogenous distribution and about 600 g soil dry
weight equivalent were filled into test vessels.


- Controls: Artificial soil moistened with deinised water without test or reference item was used as control medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution): None

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Eisenia fetida, subspecies Eisenia fetida fetida
- Source: Own breeding
- Age at test initiation (mean and range, SD): synchronized earthworm culture, not deviating more than 1 month
- Weight at test initiation (mean and range, SD): 0.44

ACCLIMATION
- Acclimation period: 2 days prior to test start
- Acclimation conditions (same as test or not): same as test
- Health during acclimation (any mortality observed): healthy

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

Removement of adults after 28 days

Test temperature:

18 – 22 °C

pH:

Application rate
[mg test item/
kg soil
dry weight] pH-value
day 0 day 56
Control 6.13 6.27
62.5 6.03 6.25
125 6.16 6.19
250 6.24 6.28
500 6.29 6.28
1000 6.45 6.27

Moisture:

Application rate
[mg / kg soil
dry weight] Soil moisture [%]
day 0 day 56
Control 28.1 35.0*
62.5 28.3 33.8*
125 28.2 33.7*
250 28.3 33.8*
500 28.7 33.1*
1000 28.1 33.7*

* Soil moisture content deviated by more than 10 %.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): round plastic boxes, ID: 15 cm, bottom surface area: 177 cm2, height: 14 cm, transparent and perforated lids enabled sufficient gas exchange and light penetration and prevented the test substrate from drying
- Amount of soil or substrate: 600 g dw
- No. of organisms per container (treatment): 10 (40)
- No. of replicates per treatment group: 4
- No. of replicates per control: 8



SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Composition (if artificial substrate): 10 % peat, 20 % kaolin, 69 % air dried quartz sand, 0.40 % calcium carbonate
- Organic carbon (%): not determined
- Maximum water holding capacity (in % dry weigth): 52.9
- CEC: not determined
- Pretreatment of soil: none
- Storage (condition, duration): 9 days after preparation at test conditions in the dark



OTHER TEST CONDITIONS
- Photoperiod: 16 h light - 8 h dark
- Light intensity: 532 +/- 94 lx

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Adult mortality, biomass, behaviour and pathological symptoms after 28 days
Reproduction (Number of juveniles) after 56 days

VEHICLE CONTROL PERFORMED: No

Nominal and measured concentrations:

Nominal application rate: 62.5 – 125 – 250 – 500 – 1000 mg test item/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

0.5 - 1 - 2 - 4 mg Carbendazim/ kg soil dw

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

170 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

250 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

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

Remarks:

groth

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

groth

Details on results:

- Mortality at end of exposure period: No significant mortality (< 10 %) of adult earthworms was observed in the control as well as at all treatment rates after 28 days of exposure to the test item.
- Changes in body weigth of live adults (% of initial weight) at end of exposure period:
At the test start the earthworms had individual weights of 0.32 - 0.59 g, which are consistent with the range recommended by the test guideline. The biomass of the adult earthworms increased throughout the first 28 days at all application rates, with no statistically significant differences (Williams Sequential t-test) compared to the control.
- No. of offspring produced: The mean number of juveniles per replicate in the control was 183. The average number of juveniles per replicate in the treated groups ranged from 144 to 186 . There was a reduction (0 – 21.5 %, compared to the control) in the number of juveniles produced across the range of test item treatments and the reproduction of the earthworms was statistically significantly different from the control at the test item concentrations 500 and 1000 mg test item/kg soil dry weight. The EC50-value was not determined since no reduction of reproduction ≥ 50 % occurred, but it exceeded 1000 mg test item/kg soil dry weight, the highest concentration tested. The EC10-value was determined to be 170 mg test item/kg soil dry weight. The coefficient of variation calculated for the reproduction of the control was 7.81 % and was thus lower than 30 %.
- Behavioural abnormalities: No pathological symptoms nor changes in the behaviour of the earthworms were observed in the control or in any of the application rates.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: 4 mg Carbendazim/kg soil dw

Reported statistics and error estimates:

Significant differences in biomass gain and reproduction were determined in comparison with the control group using Welch-t-test with Bonferroni-Holm Adjustment (biomass in [g]) and Williams Multiple Sequential t-test (biomass in [%], reproduction).
When running the test, a Normality Test and an Equal Variance Test were done first. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05. For details please refer to section 10.
The EC50-values for growth and reproduction were not calculated since no test item related reduction of body weight gain and reproduction exceeding 50 % occurred at the application rates tested. The EC10-value for reproduction was calculated using ToxRat Professional.

Mortality of Adult Earthworms in [%] after 28 Days of Exposure

Application rate	Replicate
[mg test item/kg soil dry weight]	1	2	3	4	5	6	7	8	Mean

Control	0	0	0	0	20	0	0	0	2.5
62.5	0	0	0	0					0
125	0	0	0	0					0
250	0	0	0	0					0
500	0	0	0	10					2.5
1000	0	0	0	0					0

Earthworm Behaviour and Pathological Symptoms after 28 Days of Exposure

Application rate	Replicate
[mg test item/kg soil dry weight]	Observation parameter	1	2	3	4	5	6	7	8
Control	A H	10/10 -	10/10 -	10/10 -	10/10 -	8/10 2/10	10/10 -	10/10 -	10/10 -
62.5	A H	10/10 -	10/10 -	9/10 1/10	10/10 -	–
125	A	10/10	10/10	10/10	10/10
250	A	10/10	10/10	10/10	10/10
500	A H	10/10 -	10/10 -	10/10 -	9/10 -
1000	A	10/10	10/10	10/10	10/10

A) No visible symptoms or behavioural changes H) dead

Body Weight Changes of the Adult Earthworms

Application rates [mg/kg soil dry weight]	Replicate	Mean body weights per replicate		Mean body weight gain of earthworms
		Test start	28 days	Per replicate		Mean ± SD		Sig.
		[g]	[g]	[g]	[%]	[g]	[%]
Control	1	0.45	0.60	0.15	33.3	0.16 ± 0.02	36.3 ± 4.67	–
	2	0.44	0.61	0.17	38.6
	3	0.45	0.58	0.13	28.9
	4	0.45	0.63	0.18	40.0
	5	0.44	0.58	0.14	31.8
	6	0.44	0.60	0.16	36.4
	7	0.44	0.63	0.19	43.2
	8	0.45	0.62	0.17	37.8
62.5	1	0.45	0.61	0.16	35.6	0.16 ± 0.04	37.0 ± 9.43	No
	2	0.44	0.55	0.11	25.0
	3	0.43	0.60	0.17	39.5
	4	0.44	0.65	0.21	47.7
125	1	0.43	0.64	0.21	48.8	0.18 ± 0.02	40.6 ± 5.56	No
	2	0.44	0.61	0.17	38.6
	3	0.44	0.61	0.17	38.6
	4	0.44	0.60	0.16	36.4
250	1	0.44	0.60	0.16	36.4	0.15 ± 0.01	34.1 ± 3.25	No
	2	0.44	0.59	0.15	34.1
	3	0.44	0.57	0.13	29.5
	4	0.44	0.60	0.16	36.4
500	1	0.43	0.62	0.19	44.2	0.18 ± 0.02	40.4 ± 3.72	No
	2	0.45	0.64	0.19	42.2
	3	0.45	0.61	0.16	35.6
	4	0.43	0.60	0.17	39.5
1000	1	0.44	0.65	0.21	47.7	0.18 ± 0.04	41.0 ± 8.45	No
	2	0.44	0.59	0.15	34.1
	3	0.45	0.67	0.22	48.9
	4	0.45	0.60	0.15	33.3

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, Pentaethylenehexamine, PEHA 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 adult earthworm biomass increased in the control and at all application rates without any statistically significant differences. After a further four weeks of exposure, the numbers of juvenile earthworms were significantly lower at the test item concentrations 500 and 1000 mg test item/kg soil dry weight than in the untreated control.
Thus, the No Observed Effect Concentration (NOEC) of Pentaethylenehexamine, PEHA summarizing the endpoints mortality, growth of Eisenia fetida after 4 weeks of exposure in artificial soil was determined to be ≥ 1000 mg test item/kg soil dry weight. The NOEC in terms of reproduction after 8 weeks of exposure was determined to be 250 mg/kg soil dry weight.

Executive summary:

Effects ofPentaethylenehexamine, PEHA (Batch no. P0982) on mortality, growth and the reproductive potential of the earthworm speciesEisenia fetida (Annelida, Lumbricidae) were determined according to OECD Guideline 222 (2004) from August 28^th to October 24^th, 2014 with the definitive exposure phase from August 28^th to October 22^nd/23^rd, 2014 at Dr.U.Noack-Laboratorien in D-31157 Sarstedt, Germany. The study was conducted over 8 weeks with test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight, which were mixed into artificial soil containing 10 % peat. A control using untreated artificial soil was also set up.Sexually mature earthworms were inserted into 8 control replicates and into 4 replicates per test item concentration (10 earthworms per replicate). The earthworms had an individual body weight between 0.32 and 0.59 g at the start of exposure.

After 28 days of exposure in soil, no evident earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms were observed in all treatments. Moreover, no statistically significant differences in growth (earthworm body weight gain) were observed in all test item concentrations compared to the control. Therefore, no EC_x-values in terms of mortality and growth were determined. After a further four weeks the reproduction rate (average number of juveniles produced per replicate) was 183 in the control and ranged from 144 to 186 in the test item treatment groups. Compared to the control, a statistically significant reduction of reproduction was observed at test item concentrations of 500 and 1000 mg/kg soil dry weight. Consequently, the NOEC of the test item concerning reproduction was determined to be 250 mg/kg soil dry weight. The EC₅₀- and the EC₂₀-values for reproduction and all EC_x-values in terms of mortality and growth could not be calculated, since the reduction of reproduction was below the respective thresholds. The EC₁₀-value for reproduction was determined to be 170 mg test item/kg soil dry weight.

 

All validity criteria specified by the test guideline were fulfilled.

Summary of All Observed Effects in the Tested Application Rates after 28 / 56 days

Effects	Pentaethylenehexamine,PEHA
[mg test item/kg soil dry weight]	62.5	125	250	500	1000
Mortality of adult earthworms1)	No	No	No	No	No
Reduction of growth (Body weight gain)1)	No	No	No	No	No
Reduced reproduction rate  (Number of juveniles)2)	No	No	No	Yes	Yes
Summary of all effects	No	No	No	Yes	Yes

NOEC, LOEC and EC_x-values after 28 / 56 days

Endpoint	[mg test item/kg soil dry weight]
LOECmortality,growth1)	> 1000
LOECreproduction 2)	500
NOECmortality,growth 1)	≥ 1000
NOECreproduction 2)	250
EC10, 20, 50mortality, growth 1)	> 1000
EC10reproduction 2)	170
EC20, 50reproduction 2)	> 1000

1) Assessment after 28 days of exposure

2) Assesment after 56 days of exposure

Description of key information

EC10(56 d): 170 mg/kg soil dw based on reproduction 

Key value for chemical safety assessment

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

170 mg/kg soil dw

Additional information

Effects of the substance on survival, growth and the reproductive potential of the earthworm species Eisenia fetida (Annelida, Lumbricidae) were determined according to OECD Guideline 222 (GLP). The study was conducted over 8 weeks with test item concentrations of 62.5, 125, 250, 500 and 1000 mg/kg soil dry weight, which were mixed into artificial soil containing 10 % peat. A control using untreated artificial soil was also set up. Adult earthworms (with clitellum) were inserted into 8 control replicates and into 4 replicates per test item concentration (10 earthworms per replicate).The earthworms had an individual body weight between 0.32 and 0.59 g at the start of exposure. 

After 28 days of exposure in soil, no evident earthworm mortalities, pathological symptoms or changes in the behaviour of adult earthworms were observed in all treatments. Moreover, no statistically significant differences in growth (earthworm body weight gain) were observed in all test item concentrations compared to the control. Therefore, no ECx-values in terms of mortality and growth were determined. After a further four weeks the reproduction rate (average number of juveniles produced per replicate) was 183 in the control and ranged from 144 to 186 in the test item treatment groups. Compared to the control, a statistically significant reduction of reproduction was observed at test item concentrations of 500 and 1000 mg/kg soil dry weight. Consequently, the NOEC (56 d) of the test item concerning reproduction was determined to be 250 mg/kg soil dry weight. The EC50- and the EC20-values for reproduction and all ECx-values in terms of mortality and growth could not be calculated, since the reduction of reproduction was below the respective thresholds. The EC10-value for reproduction was determined to be 170 mg test item/kg soil dry weight.