Oligomerisation products of alpha-pinene and beta-pinene

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:

From 2017-08-22 to 2018-31-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 17 march 2017

Specific details on test material used for the study:

Not applicable.

Analytical monitoring:

no

Details on sampling:

Not relevant.

Vehicle:

yes

Remarks:

Acetone

Details on preparation and application of test substrate:

A stock solution was prepared by dissolving 6562.5 mg of Terpenic Oligomers in 25 mL acetone. A dilution series was prepared as shown below. Then 8 mL of the corresponding dilutions were added to 20 g fine quartz sand. The treated sand was left for three hours in a fume hood until the solvent had evaporated and was mixed with a spoon. Then artificial soil equivalent to 2080 g dry weight was added to test item blended sand.
There were no significant deviations to the nominal target concentration (< 5%). The solvent control was treated with the same amount of acetone and sand as the test item groups and the control was left untreated. While mixing the artificial soil in a laboratory mixer for approximately 5 min the soil of each treatment group was moistened with deionised water. Each group was treated in one batch (two in the control) which was then split into the replicates.
Final concentrations in soils were ;16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg test item/kg soil

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: Annelida: Oligochaeta
- Source: Bred under standardised conditions at ibacon laboratories in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature.
- Age at test initiation: Adults. Approximately 9 months, with well-developed clitellum, age range between test individuals not differing by more than 4 weeks.
- Weight at test initiation: 306 mg to 590 mg

ACCLIMATION
- Acclimation period: 1day
- Acclimation conditions: same as test conditions

FEEDING:
Finely ground cattle manure was used as food. 5 g/container was scattered on the soil surface at day 1 after application and was moistened with 5 g deionised water; 5 g/container (moistened with 2 g deionised water) was added each week for the first 4 weeks of the experiment, when the food of the previous week had almost been consumed. If the food was not quite fully consumed, the added amount of food was adjusted to replace the visually estimated consumption. Four weeks after application, the food was mixed into the substrate following removal of the adult worms.

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

Based on OECD 222 but with reduced organic matter content.

Limit test:

no

Total exposure duration:

8 wk

Remarks:

Assessment of adult worm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult worms in treated artificial soil. Reproduction rate was assessed after additional 28 days.

Post exposure observation period:

None.

Test temperature:

Within the range of 18 °C to 22 °C

pH:

pH range at experimental start: 5.6 to 5.7
pH range at experimental end: 6.4 to 6.6

Moisture:

At experimental start: 19.3% to 20.9% (49.4% to 53.5% of the maximum water holding capacity)
At experimental end: 21.0% to 26.1% (53.9% to 67.0% of the maximum water holding capacity)

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic boxes (18.3 cm x 13.6 cm x 6 cm, tapered towards the bottom, with a soil surface of approximately 16.5 cm x 11.5 cm = 189.75 cm²) with perforated transparent lids to enable exchange of air, to minimise evaporation from the artificial soil, and to prevent the worms from escaping. Each container was filled with 602.3 g of the prepared soil (500 g dry weight plus deionised water). The height of the soil layer in the containers was approximately 5 cm.
- No. of organisms per container (treatment):10 per replicate, i.e. 80 individuals per control, and 80 individuals per solvent control, 40 individuals per test item treated group
- No. of replicates per treatment group:8 for the control, 8 for the solvent control, 4 per test item treated group

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
Based on OECD 222 but with reduced organic matter content:
- 5% Sphagnum-peat, air-dried and finely ground (<2 mm, with no visible plant remains); (Floragard, Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany)
- 20% Kaolin clay (Erbslöh, 65558 Lohrheim, Germany)
- 74.8% fine quartz-sand (F34) containing more than 50% by mass of particle size 0.05 mm to 0.2 mm; (Quarzwerke Frechen, Postfach 1780, 50207 Frechen, Germany)
- 0.2% Calcium carbonate (CaCO3) was added to adjust pH to 6.0 ± 0.5 (Merck, 64293 Darmstadt, Germany).
According to OECD 222 and EPPO (2003), 5 % of peat was used in the artificial soil considering a potential influence of the test item on bioavailability.

OTHER TEST CONDITIONS
- Photoperiod: 16 h light / 8 h dark
- Light intensity: Within the range of 400 lux to 800 lux

EFFECT PARAMETERS MEASURED :
- Mortality: Number of dead adult earthworms at day 28 after application (including any missing ones)
- Behavioural and Morphological Abnormalities: Number of affected adult earthworms (e.g. lack of movement, rigidity) at day 28 after application
- Food Given: Cumulative amount of food added to each test container during the test period
- Mean Body Weight: Body weights were determined at start (day 0) and 28 days after application
- Reproduction: Number of juveniles 56 days after application

VEHICLE CONTROL PERFORMED: yes

TEST CONCENTRATIONS
- Main test: 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg test item/kg soil
- Range finding test: yes
- Test concentrations (range-finder): 0.1, 1, 10, 100, 1000 mg/kg

Nominal and measured concentrations:

Control, Solvent Control, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg (nominal) Terpenic Oligomers/kg soil (dw).

Reference substance (positive control):

yes

Remarks:

Carbendazim

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 on result:

other: Results compared to controls

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 on result:

other: Results compared to controls

Duration:

28 h

Dose descriptor:

NOEC

Effect conc.:

308.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Weight

Remarks on result:

other: Results compared to the pooled control

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

555.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: weight

Remarks on result:

other: Results compared to the pooled control

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

308.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Results compared to the pooled control

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

555.6 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Results compared to the pooled control

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

139.8 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

95% CL: 20.4-251.5

Key result

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

1 336.1 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other:

Remarks:

95% CL: 795.1-6514.4

Details on results:

A slight mortality of 2.5% was found at the concentration of 29.4 mg test item/kg soil which was not statistically significantly different compared to either control – control and solvent control -, where 0% of the worms died, respectively (Fisher’s Exact Test, a = 0.05, one-sided greater).
The body weight changes of the earthworms after 4 weeks exposure to Terpenic Oligomers were not statistically significant up to concentration of 308.6 mg test item/kg soil but in the test item concentration of 555.6 mg test item/kg soil and above weight changes were statistically significantly reduced compared to the pooled control group (Williams t-test, a =0.05, one-sided smaller).
No statistically significant effects on reproduction were observed up to concentration of 308.6 mg test item/kg soil but in the test item concentration of 555.6 mg test item/kg soil and above reproduction was statistically significantly reduced compared to the pooled control group (Bonferroni-Welch t-test, a =0.05, one-sided smaller). No behavioural abnormalities were observed in any of the treatment groups and the feeding activity in all the treated groups was comparable to the control.

See table below.

Results with reference substance (positive control):

In the most recent test with the reference item Carbendazim 600 g/L SC (performed under ibacon Study Number 105683022 from July to September 2017), there were statistically significant effects on reproduction at a concentration of 1.44 mg carbendazim/kg soil and higher, which is in line with the guideline OECD 222 (effects should be observed between 1 and 5 mg carbendazim/kg soil). The EC50 for reproduction was calculated as 1.47 mg carbendazim/kg soil.

Reported statistics and error estimates:

See table below.

Effect of Terpenic Oligomers on earthworms (Eisenia andrei) in a 56-day reproduction study

[mg test item/kg soil dry weight]	Control		Solvent Control	Pooled Control	16.3		29.4	52.9	95.3	171.5		308.6	555.6	1000
Mortality (day 28) [%]	0		-	-	0		2.5	0	0	0		0	0	0
Statistical Significance^1)4)	-		-	-	n.s.		n.s.	n.s.	n.s.	n.s.		n.s.	n.s.	n.s.
Mortality (day 28) [%]	-		0	-	0		2.5	0	0	0		0	0	0
Statistical Significance^1) 5)	-		-	-	n.s.		n.s.	n.s.	n.s.	n.s.		n.s.	n.s.	n.s.
Body weight change (day 28) [%]	50.1		46.7	48.4	41.7		45.2	43.2	48.5	44.3		43.1	37.4	36.0
Statistical Significance^2) 6)	-		n.s.^7)	-	n.s.		n.s.	n.s.	n.s.	n.s.		n.s.	*	*
Mean No. of juveniles (day 56)	202		226	214	209		223	211	194	191		190	143	118
Statistical Significance^3) 6)	-		n.s.^7)	-	n.s.		n.s.	n.s.	n.s.	n.s.		n.s.	*	*
Reproduction in [%] of pooled control (day 56)	-		-	-	97.4		104	98.5	90.4	89.1		88.6	66.8	55.0
		Endpoints [mg test item/kg soil dry weight]
NOEC (day 28 mortality)^4)5)		1000
LOEC (day 28 mortality)^4)5)		>1000
NOEC (day 28 weight)^6)		308.6
LOEC (day 28 weight)^6)		555.6
NOEC (day 56 reproduction)^6)		308.6
LOEC (day 56 reproduction)^6)		555.6
EC Values(reproduction)^8^)		EC10				EC20					EC50
		139.8				303.4					1336.1
95% confidence limits		20.4 – 251.5				117.4 – 469.1					795.1 – 6514.4

The results represent rounded values calculated on the exact raw data.
- = not applicable    
n.s. = not significantly different compared to the control (pooled control: weight changes and reproduction)
* = significantly different compared to the pooled control (pooled control: weight changes and reproduction)
¹⁾Fisher’s Exact Test,a= 0.05, one-sided greater                                      
²⁾Williams t-test,a= 0.05, one-sided smaller
³⁾Bonferroni-Welch t-test,a= 0.05, one-sided smaller
⁴⁾Compared to the control
⁵⁾Compared to the solvent control
⁶⁾Compared to the pooled control
⁷⁾Student t-test,a= 0.05, two-sided
⁸⁾Probit analysis     

Validity criteria

Control mortality: Should not exceed 10% over initial 4-week test period. ontrol mortality was 0% and so this validity criterion was met.

Reproduction of control: Should be³30 worms per replicate container. The number of juvenile worms per replicate was 146 to 273 ans so this criterion was met.

Coefficient of variation of Reproduction incontrol : Should not exceed 30%. It was 18.7% and so this validity criterion was met.

Validity criteria fulfilled:

yes

Conclusions:

In an earthworm reproduction and growth study with Terpenic Oligomers the No Observed Effect Concentration (NOEC) for mortality was determined to be the concentration of 1000 mg test item/kg soil and the Lowest Observed Effect Concentration (LOEC) for mortality was estimated to be >1000 mg test item/kg soil.
The NOEC for weight changes and reproduction of the earthworm Eisenia andrei was determined to be the concentration of 308.6 mg test item/kg soil and the LOEC for weight changes and reproduction was determined to be the concentration of 555.6 mg test item/kg soil.
The EC10 was determined to be 139.8 mg test item/kg soil (95% confidence limits of 20.4 to 251.5 mg test item/kg soil), the EC20 was determined to be 303.4 mg test item/kg soil (95% confidence limits of 117.4 to 469.1 mg test item /kg soil) and the EC50 was determined to be 1336.1 mg test item/kg soil (95% confidence limits of 795.1 to 6514.4 mg test item/kg soil).

Executive summary:

The effect of test item Terpenic Oligomers to earthworm reproduction and growth has been investigated during an experimental GLP study following OECD Guideline 222.

Effects to adult worms were determinated during a 56-day test in treated artificial soil prepared according to OECD Guideline 222 (5% peat only). Different concentrations of the test item were tested within 10 soil treatment groups (8 test item concentrations, control, solvent control). 4 replicates were prepared for test item treatments and 8 replicates for the controls. Assessment of adult worm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult worms in treated artificial soil. Reproduction rate (number of offspring) was assessed after additional 28 days (assessed 56 days after application).

The No Observed Effect Concentration (NOEC) for mortality was determined to be the concentration of 1000 mg test item/kg soil and the Lowest Observed Effect Concentration (LOEC) for mortality was estimated to be >1000 mg test item/kg soil.

The NOEC for weight changes and reproduction of the earthworm, Eisenia andrei, was determined to be the concentration of 308.6 mg test item/kg soil and the LOEC for weight changes and reproduction was determined to be the concentration of 555.6 mg test item/kg soil.

The EC₁₀was determined to be 139.8 mg test item/kg soil (95% confidence limits of 20.4 to 251.5 mg test item/kg soil), the EC₂₀ was determined to be 303.4 mg test item/kg soil (95% confidence limits of 117.4 to 469.1 mg test item /kg soil) and the EC₅₀ was determined to be 1336.1 mg test item/kg soil (95% confidence limits of 795.1 to 6514.4 mg test item /kg soil).

All study validity criteria were met and therefore the study is considered as valid.

Description of key information

Under experimental conditions and based on nominal concentrations, the 56h-EC₅₀ and the NOEC of Terpenic oligomers are of 1336.1 and 308.6 mg test item/kg soil dw, respectively.

Several biologic parameters were measured (mortality, weight change, feeding activity and reproduction rate). EC₅₀ and NOEC values were obtained by assessing the more sensitive parameter, effect to reproduction.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 336.1 mg/kg soil dw

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

308.6 mg/kg soil dw

Additional information

Assessment of adult worm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult worms in treated artificial soil. Reproduction rate (number of offspring) was assessed after additional 28 days (assessed 56 days after application).

The No Observed Effect Concentration (NOEC) for mortality was determined to be the concentration of 1000 mg test item/kg soil and the Lowest Observed Effect Concentration (LOEC) for mortality was estimated to be >1000 mg test item/kg soil.

The NOEC for weight changes and reproduction of the earthworm, Eisenia andrei, was determined to be the concentration of 308.6 mg test item/kg soil and the LOEC for weight changes and reproduction was determined to be the concentration of 555.6 mg test item/kg soil.

The EC₁₀was determined to be 139.8 mg test item/kg soil (95% confidence limits of 20.4 to 251.5 mg test item/kg soil), the EC₂₀ was determined to be 303.4 mg test item/kg soil (95% confidence limits of 117.4 to 469.1 mg test item /kg soil) and the EC₅₀was determined to be 1336.1 mg test item/kg soil (95% confidence limits of 795.1 to 6514.4 mg test item /kg soil).