Oligomerisation products of alpha-pinene and beta-pinene

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-26 to 2018-XX-XX

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to the OECD 225 guideline and under GLP compliance.

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 225 (Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment)

Deviations:

yes

Remarks:

There was one deviation from the study plan concerning some temperature values, that did not affect the integrity of the study.

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on 2017-01-10

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Single sample for analysis were taken from the controls and from all test concentrations (overlying water + bulk sediment) at the start (just before adding the test organisms), at day 7 and at the end of the test.

Vehicle:

yes

Remarks:

acetone

Details on sediment and application:

ARTIFICIAL SEDIMENT
The artificial sediment was prepared the day before the preparation of the spiked sediment, based on the recommendations of OECD Guideline 225 (see "any other information on material and methods incl. tables").
The pH of the final mixture was adjusted to 6.5 with CaCO3. Samples of the sediment were taken to determine the dry weight and the organic carbon content (in accordance with ISO 17025): the percentage of dry matter was around 70% and the TOC contents was approx. 2% (19.14 g.kg-1 of dry matter). The reconstituted sediment was stored at 5+/-3°C before use the next day.

PREPARATION OF THE SPIKED SEDIMENT
A stock solution of the test item was prepared in acetone at 20 g.L-1. The stock solution was thereafter diluted with acetone to prepare the test solutions. Then, the sediment was spiked in bulk for each concentration level. For this purpose, each of the test solutions was mixed with quartz sand in open glass dish: a volume of 0.25 mL of test solution per g of sand was employed in order to soak the quartz sand completely. Thereafter, the solvent was evaporated to dryness for approx. 1-2 hours under fume hood. Then the coated sand was mixed by hand with the suitable amount of artificial sediment of the corresponding concentration level to achieve the desired nominal concentration levels (in mg.kg-1 dry weight of sediment). The spiked sediment was then distributed to the replicate test vessels for each treatment, and topped with test water (sediment-water ratio of approx. 1:4).

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

- Species: Lumbriculus variegatus (Müller)
- Origin: ECT Oekotoxikologie GmbH - Böttgerstrasse 2-14 - 65439 Flörsheim am Main - Germany, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: This worm species is tolerant to a wide range of sediment types, and is widely used for sediment toxicity and bioaccumulation testing, according to OECD Guideline 225.
- Validity of batch: Animals from a healthy stock, and free from observable diseases and abnormalities.

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

No data

Hardness:

Hardness was approximately 300 mg CaCO3.L-1 at the start of the test

Test temperature:

The temperature of the test medium recorded continuously in the vessel (exclusively dedicated to temperature control) next to the test vessels was situated between 19.3 and 25.1°C throughout the test (average value: 21.0°C).
Requirements: 20°C ± 2°C; should not, if possible, vary by more than 1°C within these limits daily

pH:

7.70 - 8.82
Requirements: between 6.0 and 9.0.

Dissolved oxygen:

5.15 - 8.50
Requirements: dissolved [02] in overlying water ≥ 30% of air saturation value at test temperature during the test.

Ammonia:

Ammonium content was not higher than 1 mg NH4+.L-1 throughout the test

Nominal and measured concentrations:

Nominal: Control, Solvent Control, 31.25, 62.50, 125.00, 250.00 and 500.00 mg/kg

Details on test conditions:

HOLDING
- Breeding conditions: Worms were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of Lumbriculus variegatus was performed in glass aquaria containing a layer of quartz sand (approx. 1-2cm depth) and test water (the water body was gently aerated via a pasteur pipette). Worms were fed at least twice a week with a suspension (50 mg.mL-1) of TetraMin or Urtica dioica (stinging nettle). The water was changed at least once a week.
- Synchronisation: 14 days before the start of exposure, worms were artificially fragmented (= synchronisation, to minimise uncontrolled reproduction and regeneration). Adult worms, preferably not showing signs of recent morphallaxis, were selected for synchronisation. These worms were placed onto a glass slide on a drop of test water, and were dissected in the median body region with a scalpel. The posterior ends were then left to regenerate new heads in all-glass vessels containing quartz sand and test water until the start of exposure, under similar temperature and light conditions as used in the test. Worms were fed once as soon as they started to burrow in the sand, or 7 days after dissection, with a suspension (50 mg.mL-1) of TetraMin or Urtica dioica. After regenerating, intact complete worms, which were actively swimming or crawling upon a gentle mechanical stimulus, were used for the test.

TEST CONCENTRATIONS
- Test item: Based on the results of a range-finding test, test solutions used in the definitive test were prepared to obtain the following nominal test concentrations (spaced by a factor of 2.00): 31.25, 62.50, 125.00, 250.00 and 500.00 mg test item.kg-1 dry weight of sediment, in agreement with the Study Monitor.
- Controls: Containing all constituents but without test item, and treated in the same way as the test item solutions. One control series containing acetone* (solvent control) at the level used in the test concentrations was also prepared.
- *Solvent: Acetone (CAS No. 67-64-1; CARLO ERBA REAGENTS, batch No. D6N043097A) with density at 20°C of 0.79 g/cm3, and purity ≥ 99.8%.
The selection of the solvent was based on the experience from the Sponsor on handling the test item. The range-finding test revealed that a solvent was necessary for the preparation of test solution.

RANGE-FINDING TEST
Ten worms per concentration were exposed to the nominal concentrations of 1, 10, 100 and 1000 mg.kg-1 (in duplicate) and to controls (in triplicate) with and without solvent. Due to an error during the preparation of the spiked sediment, the nominal concentrations were 0.1, 1.0, 10.0 and 100.0 mg.kg-1 instead.

TEST PROCEDURE AND CONDITIONS
- Test vessels: All-glass beakers of approximately 250 mL capacity and with diameter 6 cm, covered with parafilm. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Test (overlying) water: Reconstituted water, as prescribed by OECD Guideline 203.
- Number of animals: 10 synchronised worms per replicate were introduced in each test vessel at the beginning of the test
- Loading: Each test vessel contained a layer of spiked sediment topped with test water (sediment-water ratio of approx. 1:4).
- Number of replicates: 4 replicates per test concentration, and 6 replicates for the controls (with and without solvent). Additional replicates were prepared and sampled for chemical analyses.
- Feeding: Since food was added to the sediment prior to application of the test substance, the worms were not fed additionally during the test.
- Test environment: Controlled environment cabinet (20°C ± 2°C).
- Light regime/intensity: 16h light : 8 h dark; light intensity was kept low (between 100-500 lux).
- Aeration: The overlying water of the test vessels was gently aerated via a pasteur pipette positioned approx. 2 cm above the sediment surface so as to minimise perturbation of the sediment. Air supply was controlled and adjusted at least once daily on workdays.
- Equilibration period: Once the spiked sediment was prepared, distributed to the replicate test vessels, and topped with the test water, an equilibration period was done for 4 days under similar temperature, light and aeration conditions as used in the test.
- Introduction of animals: Worms were introduced into test vessels at the end of the equilibration period.

MEASUREMENTS AND RECORDINGS
- Biological observations: The test vessels were observed during the exposure in order to assess visually any behavioural differences in the worms compared with the controls. Water lost due to evaporation from the test vessels was topped up with deionised water.
- Total number of worms : At the end of the test, each replicate was examined. The worms were sieved from the sediment, rinsed with water and the total number of surviving worms per replicate was determined.
- Total dry weight of worms: Immediately after counting, the living worms found in each replicate were transferred to dried, pre-weighed and labelled weigh pans (one per replicate), and killed using a drop of ethanol per weigh pan. The weigh pans were placed in a drying oven at 100 ± 5°C to dry overnight, after which they were weighed and worm dry weight was determined.
- Dissolved O2, pH: Measured in one replicate of each treatment and control once a week.
- Temperature of test water : Measured continuously in a temperature control vessel next to the test vessels, over the study period, beginning at the start of the test. Moreover, temperature was recorded in one replicate of each treatment and control once per week, during pH and dissolved O2 determinations.
- Total water hardness: Measured in one replicate of each treatment and control at the start and the end of the exposure period.
- Ammonium content: Measured in one replicate of the controls and one replicate of each treatment at the start of the exposure period, and subsequently 3 times a week.
- Light intensity: Light intensity was measured once, at the start of the test.

Reference substance (positive control):

no

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

1.4 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

total number of worms

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

19.5 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

239 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

total number of worms

Duration:

28 d

Dose descriptor:

EC50

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

not determinable

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

62.5 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

total number of worms

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 500 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

125 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

total number of worms

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

> 500 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

BIOLOGICAL RESULTS
See tables 6.2/2 and 6.2/3 in the section "any other information on results incl. tables".

ANALYTICAL RESULTS
To be completed

WATER QUALITY PARAMETERS AND ENVIRONMENTAL CONDITIONS
pH and dissolved oxygen concentrations respected the requirements.
Hardness was approximately 300 mg CaCO3.L-1 at the start of the test and ammonium content was not higher than 1 mg NH4+.L-1 throughout the test.
The light intensity was situated between 124 and 466 lux, which remained within the ranges prescribed (between 100-500 lux).
The temperature of the test medium recorded continuously in the vessel (exclusively dedicated to temperature control) next to the test vessels was situated between 19.3 and 25.1°C throughout the test (average value: 21.0°C), which was higher than the requirements (20°C ± 2°C; should not, if possible, vary by more than 1°C within these limits daily). This minor deviation was considered not to affect the results of the test as no impact was observed on the controls throughout the duration of the test. Moreover, temperatures recorded in test vessels during pH and dissolved O2 determinations complied with the requirements (20°C ± 2°C; not varying by more than 1°C within these limits daily).

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

The evaluation of the concentration-effect-relationships and the calculations of effect concentrations were determined by the computer program ToxRat. ECx values are given for information and should be taken cautiously, since a significant lack of fit was found by the software.

6.2/2: Total worm number at the end of the test.

 

Replicate	Nominal concentration(mg.kg^-1)
	Control	Solvent control	31.25	62.50	125.00	250.00	500.00
1	20	21	30	31	0	16	13
2	24	17	0	11	0	13	18
3	36	27	13	17	6	5	2
4	15	12	24	0	25	13	10
5	33	19	-	-	-		-
6	20	18
Mean	24.7	19.0	16.8	14.8	7.8	11.8	10.8
Std. Dev.	8.19	4.94	13.20	12.92	11.84	4.72	6.70
CV	33.2	26.0	78.8	87.6	152.8	40.1	62.3
% Inh. (in comparison with the control)	-	/	32.1	40.2	68.6	52.4	56.4
% Inh. (in comparison with the solvent control)	/	-	11.8	22.4	59.2	38.2	43.4

% Inh.: %Reduction of worm number by the test item

 

Table 6.2/3: Biomass (dry weight in mg) at the end of the test.

 

Replicate	Nominal concentration(mg.kg^-1)
	Control	Solvent control	31.25	62.50	125.00	250.00	500.00
1	14.05	12.64	19.79	20.64	-	11.93	11.35
2	25.27	13.41	-	12.15	-	12.1	14.53
3	30.46	18.18	5.72	10.20	3.75	6.47	2.25
4	8.71	7.50	14.02	-	18.52	11.98	10.99
5	22.12	12.43	-	-	-		-
6	15.88	9.77
Mean	19.42	12.32	13.18	14.33	11.14	10.62	9.78
Std. Dev.	7.99	3.62	7.07	5.55	10.44	2.77	5.27
CV	41.2	7.07	53.7	38.7	93.8	26.1	53.8
% Inh. (in comparison with the control)	-	/	32.1	26.2	42.6	45.3	49.6
% Inh. (in comparison with the solvent control)	/	-	-6.9	-16.3	9.6	13.8	20.6

% Inh.: %Reduction of weight caused by the test item

Validity criteria of the study

- Number of worms in controls

The average number of living worms per replicate increased by a factor of 2.5 and 1.9 in the control without solvent and in the solvent control, respectively.

- Overlying water

The pH of the overlying water was between 6 and 9 throughout the test.

The oxygen concentration in the overlying waterwas at least30% of air saturation value (ASV)throughout the test.

Validity criteria fulfilled:

yes

Conclusions:

The effects of a chronic exposure to the test item on the oligochaete Lumbriculus variegatus was investigated in this study. The evaluation was based on nominal concentrations. Under the experimental conditions and after 28 days, the NOEC for parameter " total number of surviving worms" was 62.5 mg/kg and for the parameter biomass (dry weight), the NOEC was determined to be at least 500 mg/kg.

Executive summary:

A study was performed to assess the effects of a chronic exposure to test item TERPENIC OLIGOMERS on sediment-dwelling aquatic oligochaete Lumbriculus variegatus. The followed method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 225, "Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment" and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures”.

The test animals were exposed to the test item over a range of nominal concentrations of 31.25, 62.50, 125.00, 250.00 and 500.00 mg test item/kg dry sediment, and to untreated controls (with and without solvent). The worms were exposed to the sediment-water systems for a total period of 28 days. Each treatment group consisted of 4 replicates (10 synchronised worms per replicate), and 6 replicates for the controls. Effects on Lumbriculus variegatus were assessed at the end of the test by counting the total number of living worms per replicate and by determining their dry weight, in comparison with controls. The concentrations of the test item were determined by chemical analyses at the start, at day 7 and at the end of the test.

The effect of the test item on Lumbriculus variegatus after 28 days of exposure was as follows:

	Worm number (mg test item.kg^-1)	Biomass (dry weight) (mg test item.kg^-1)
EC10	1.4	19.5
EC20	8.1	79.4
EC50	239.0	n.d.
LOEC	125.0	> 500.0
NOEC	62.5	>= 500.0

n.d.: not determined due to mathematical reasons or inappropriate data

The software ToxRat® Professional was used for the determination of the critical effect and threshold concentrations.

The evaluation was based on nominal concentrations. Therefore, after 28 days, the NOEC for parameter "total number of surviving worms" was 62.5 mg/kg and for parameter "biomass" (dry weight), the NOEC was determined to be at least 500 mg/kg.

Description of key information

The effects of a chronic exposure to the test item on oligochaete Lumbriculus variegatus was investigated.

After 28 days, the NOEC for parameter "total number of surviving worms" was 62.5 mg/kg and for parameter "biomass" (dry weight), the NOEC was determined to be at least 500 mg/kg.

Additional information

A study was performed to assess the effects of a chronic exposure to test item TERPENIC OLIGOMERS on sediment-dwelling aquatic oligochaeteLumbriculus variegatus. The followed method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 225, "Sediment-WaterLumbriculusToxicity Test Using Spiked Sediment" and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures”.

The test animalswere exposed to the test item over a range of nominal concentrations of31.25, 62.50, 125.00, 250.00 and 500.00 mg test item/kgdry sediment,and to untreated controls (with and without solvent). The worms were exposed to the sediment-water systems for a total period of 28 days. Each treatment group consisted of 4 replicates (10 synchronised worms per replicate), and 6 replicates for the controls.Effects on Lumbriculus variegatuswere assessed at the end of the test by counting the total number of living worms per replicate and by determining their dry weight, in comparison with controls. The concentrations of the test item were determined by chemical analysesat the start, at day 7 and at the end of the test.

The evaluation was based on nominal concentrations. The result can be used as key value for chemical safety assessment.