Oligomerisation products of alpha-pinene and beta-pinene

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-11-30 to 2018-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to the OECD 211 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 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

There were two deviations from the study plan (concerning some pH and temperature values). These deviations did not affect the integrity of the study.

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

signed on 2017-01-10

Analytical monitoring:

yes

Details on sampling:

Single samples for analysis were taken from the control and from all test concentrations three times a week at renewal of the test media. Samples for fresh solutions were taken from each treatment prior to addition of the daphnids and the suspension of algal cells. Samples for old solutions were centrifuged at 500 g for 5 min (in order to pellet algal cells) before sampling of the supernatant.

Vehicle:

yes

Details on test solutions:

PREPARATION OF WAFs
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace). The loading rates of the test item were weighed in glass vials and resuspended in acetone (the volume was adapted to each loading rate). Then, the test item suspensions in acetone were briefly vortexed (and sonicated for the preparation at day 0 and 3 only) to obtain homogeneous suspensions, and thereafter a specific volume (corresponding to a (limit) solvent concentration of 100 mg.L-1) of each preparation was carefully added to the surface of test water contained in the mixing vessels that were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 +/- 2 hours of gentle stirring (except for the renewal on Mondays where stirring was carried out throughout the weekend) in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were directly added into test vessels (with minimum headspace). After filling and introduction of daphnids the test vessels were sealed immediately. No small bubbles were observed in the test bottles. The test solution was observed to be clear at all concentrations, however this is not interpreted as an evidence of a true solution and absence of non-dissolved form of the test item. The test was carried out without adjustment of the pH.
At each renewal, the entire procedure was repeated when preparing fresh test solutions.


CONTROLS
Test water without test item but treated in the same way as the test item solutions. One control series containing acetone* (solvent control) at the level used in the test loading rates with solvent was also prepared.

Test organisms (species):

Daphnia magna

Details on test organisms:

- Species: Daphnia magna (Straus), clone 5
- Sex: Female
- Origin: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Validity of batch: Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at test start: < 24 hours old
- Breeding conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed at least once a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

No data

Post exposure observation period:

No data

Hardness:

The total water hardness was approximately 250 mg.L-1 (as CaCO3).

Test temperature:

Requirements: 20 °C ± 2 °C and should not, if possible, vary by more than 2°C within these limits daily

pH:

Requirements: pH: 6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

Requirements: ≥ 3 mg.L-1 at the beginning and during the test

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

Nominal: 10, 32, 100, 316 and 1000 µg/L (loading rates)

Details on test conditions:

TEST PROCEDURE AND CONDITIONS
- Test type: Semi-static and closed conditions, with three media changes per week; at each renewal, a new series of clean and sterilised test vessels was used.
- Test vessels: All-glass sealed with screw cap test bottles (air-tight) of approximately 100 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
- Test water: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 211.
- Number of animals: 10 animals individually held at each test loading rate and control series
- Loading: 1 daphnid per test vessel each completely filled with test solution and with headspace limited as much as possible
- Feeding: Daphnids were fed every working day (5 days a week) with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1.
- Test environment: Controlled environment cabinet (20°C ± 2°C)
- Light regime: 16h light : 8h dark with a light intensity not exceeding 1000 - 1500 lux.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Introduction of daphnids: Daphnids were introduced into the test medium immediately after filling the test bottles with test solutions and algal cells.

MEASUREMENTS AND RECORDINGS
- Offspring: The living offspring produced by each parent animal were removed and counted three times a week (i.e. at each renewal) from the appearance of the first brood. Only the number of living offspring was counted, but the presence of aborted eggs or dead offspring was also recorded.
- Mortality: Mortality among the parent animals was recorded daily, at the same times as offspring were counted.
- Individual length of adults: At the end of the test the length of the adults was measured by microscopy using an ocular micrometer calibrated with a stage micrometer.
- Dissolved O2, hardness, pH: Measured three times a week, in fresh and old media, in a single replicate for each treatment group.
- Temperature of medium: Measured continuously in a vessel next to the test vessels, over the study period, beginning at the start of the test.
Moreover, temperature was recorded in the control and all test concentrations (in a single replicate for each treatment group) during the pH and dissolved O2 determinations.
- Light intensity: Light intensity was measured once a week.

TEST CONCENTRATIONS
- Test item: Based on results of an acute toxicity test on D. magna (LPL Project D17-011), test solutions used in the definitive test will be prepared to obtain the following loading rates (spaced by a factor of 3.16): 10, 32, 100, 316 and 1000 µg/L.
- *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 preliminary solubility trial revealed that a solvent was necessary for the preparation of test solution; in absence of solvent, the test item was not detected at any loading rate.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

EL10

Effect conc.:

214.9 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

reproduction

Remarks on result:

other: effects are observed at levels far above the test item water solubility

Key result

Duration:

21 d

Dose descriptor:

EL50

Effect conc.:

368.9 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

reproduction

Remarks on result:

other: effects are observed at levels far above the test item water solubility

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

1 000 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

reproduction

Remarks on result:

other: effects are observed at levels far above the test item water solubility

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

316 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

loading rate of WAF

Basis for effect:

reproduction

Remarks on result:

other: effects are observed at levels far above the test item water solubility

Details on results:

BIOLOGICAL RESULTS:
See results above and tables 6.1.4/1 to 6.1.4/4 in the section "any other information on results incl. tables"

ANALYTICAL RESULTS
Due to the very low solubility of the test item in test water, it was not possible to quantify and detect the presence of test item in the lowest loading rates, despite the use of acetone for the preparation of WAFs. The test item was detected in the highest loading rates, and even quantified (> LOQ) at 316 and 1000 µg.L-1, but since control solutions were outside the tolerance limits those values cannot be considered as valid. Besides, given the complex nature of the WAF and since the test item was an UVCB substance, the results were based on nominal loading rates.

WATER QUALITY PARAMETERS AND ENVIRONMENTAL CONDITIONS
pH and oxygen concentrations remained within the limits prescribed. However, some pH values (in the control and the loading rates of 32 and 100 µg.L-1) at day 3old were slightly higher than 9.0. This was not considered to have affected the integrity of the study since no adverse effect was observed at this time in the different treatments. Hardness was above 140 mg CaCO3.L-1 as recommended.
The mean light intensity was 580 lux (range: 551-617 lux), which remained within the ranges prescribed by the study plan (not exceeding 1000-1500 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 19.2 and 21.5°C throughout the test (average value: 20.9°C), which was slightly higher than the requirements as laid down in the study plan (20 °C ± 2 °C and should not, if possible, vary by more than 2°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 before and after each renewal complied with the requirements.

Results with reference substance (positive control):

N/A

Validity criteria

Mortality: The mortality of the parent animals did not exceed 20 % at the end of the test in the controls (0%).

Offspring: The mean number of live offspring produced per parent animal surviving at the end of the test in the control without solvent was 69.6, and 71.6 in the solvent control, and so ≥ 60.

Thus all validity criteria for the performance of the controls have been fulfilled in the present study.

Table 6.1.4/1: Cumulative mortality of parental animals during the test.

  

	Nominal concentration(µg test item.L^-1)*
Day	Control	Solvent control	10	32	100	316	1000
1	0	0	0	0	0	0	0
2	0	0	0	0	0	0	0
3	0	0	0	0	0	0	0
4	0	0	0	1	0	2	8
5	0	0	0	1	0	3	8
6	0	0	0	1	0	3	8
7	0	0	0	1	0	3	8
8	0	0	0	1	0	3	8
9	0	0	0	1	0	3	8
10	0	0	0	1	0	3	9
11	0	0	0	1	0	3	10
12	0	0	0	1	0	3	10
13	0	0	0	1	0	3	10
14	0	0	0	1	0	3	10
15	0	0	0	1	0	3	10
16	0	0	0	1	0	3	10
17	0	0	0	1	0	3	10
18	0	0	0	1	0	3	10
19	0	0	0	1	0	3	10
20	0	0	0	1	0	3	10
21	0	0	0	1	0	3	10
Mortality %	0	0	0	10	0	30	100

* WAF prepared at the given loading rate.

 

 

Table 6.1.4/2: Cumulative offspring at the end of the test per introduced parent.

 

Replicate	Nominal concentration(µg test item.L^-1)*
	Control	Solvent control	10	32	100	316	1000
1	70	66.0	68	74	55	44	0
2	67	70.0	73	82	89	80	0
3	57	65.0	69	67	63	0	0
4	77	62.0	70	64	77	0	0
5	64	79.0	70	68	79	69	0
6	70	64.0	71	81	66	63	0
7	91	74.0	64	0	56	58	8
8	74	82.0	73	80	70	0	0
9	68	73.0	75	63	73	61	0
10	58	81.0	59	72	75	69	0
Mean	69.6	71.6	69.2	65.1	70.3	44.4	0.8
Std. Dev	9.81	7.35	4.71	23.90	10.59	31.97	2.53
n	10	10	10	10	10	10	10
CV	14.1	10.3	6.8	36.7	15.1	72.0	316.2

* WAF prepared at the given loading rate.

Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

 

Table 6.1.4/3: Cumulative offspring per survived parent at the end of the test.

 

Replicate	Nominal concentration(µg test item.L^-1)*
	Control	Solvent control	10	32	100	316	1000
1	70	66.0	68	74	55	44	-
2	67	70.0	73	82	89	80	-
3	57	65.0	69	67	63	-	-
4	77	62.0	70	64	77	-	-
5	64	79.0	70	68	79	69	-
6	70	64.0	71	81	66	63	-
7	91	74.0	64	-	56	58	-
8	74	82.0	73	80	70	-	-
9	68	73.0	75	63	73	61	-
10	58	81.0	59	72	75	69	-
Mean	69.6	71.6	69.2	72.3	70.3	63.4	-
Std. Dev	9.81	7.35	4.71	7.37	10.59	11.18	-
n	10	10	10	9	10	7	0
CV	14.1	10.3	6.8	10.2	15.1	17.6	-

* WAF prepared at the given loading rate.

Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

        

 

Table 6.1.4/4: Parental Daphnia magna lengths after 21 days (mm).

 

Replicate	Nominal concentration(µg test item.L^-1)*
	Control	Solvent control	10	32	100	316	1000
1	3.75	3.85	3.75	3.55	3.50	3.00	-
2	3.75	3.75	3.75	3.85	3.50	3.50	-
3	3.75	3.75	3.75	3.85	3.50	-	-
4	3.75	3.50	3.75	3.50	3.50	-	-
5	3.75	3.75	3.75	3.50	3.75	3.50	-
6	3.75	3.75	3.60	3.90	3.50	3.25	-
7	3.75	3.75	3.80	-	3.35	3.00	-
8	3.75	3.75	3.85	4.00	3.50	-	-
9	3.75	3.85	3.65	3.75	3.50	3.50	-
10	3.75	4.00	3.50	3.50	3.75	3.50	-
Mean	3.75	3.77	3.71	3.71	3.53	3.32	-
Std. Dev	0.00	0.13	0.10	0.20	0.12	0.24	-
n	10	10	10	9	10	7	0
CV	0.0	3.3	2.8	5.4	3.5	7.2	-

* WAF prepared at the given loading rate.

Std. Dev.: standard deviation; n: number of replicates; CV: coefficient of variation

Validity criteria fulfilled:

yes

Conclusions:

The effects of a chronic exposure to test item TERPENIC OLIGOMERS on the reproduction of Daphnia magna was investigated in this study. The evaluation was based on nominal loading rates. After 21 days, the NOELR value determined for the most sensitive endpoint cumulative number of offspring (316 µg/L) was at levels far above the water solubility of the test item and is not interpreted as pelagic toxicity.

Executive summary:

A study was performed to assess the effects of a chronic exposure to test item TERPENIC OLIGOMERS on the reproduction of freshwater crustacean Daphnia magna. The followed method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 211, "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Daphnids were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 10, 32, 100, 316 and 1000 µg/L, and to an untreated control and a solvent control for a total period of 21 days in closed vessels. Each treatment group consisted of 10 replicates each of one Daphnia magna (one daphnia per replicate). The test media was renewed three times a week and the daphnids were fed five days a week. Effects on reproductive performance were investigated by counting the living offspring produced by each parent animal every day. Mortality among the parent animals was also recorded. At the end of the test the length of the adults was measured by microscopy. The concentrations of the test item were determined by chemical analyses three times a week at renewal of the test media.

Due to the very low solubility of the test item in water, it was not possible to quantify and detect the presence of the test item in the lowest loading rates, despite the use of acetone for the preparation of WAFs. Nevertheless, the test item was detected in the highest loading rates, and even quantified (> LOQ) at 316 and 1000 µg/L, but since control solutions were outside the tolerance limits those values cannot be considered as valid. Given the complex nature of the WAF and since the test item was a UVCB substance, the results were based on nominal loading rates.

The effect of the test item on the reproduction of Daphnia magna after 21 days of exposure was as follows:

Critical effect and threshold concentration on cumulative offspring per introduced parent after 21 days (µg test item.L^-1)		95% confidence limits (µg test item.L^-1)
ELR10	214.9	84.104 – 549.108
ELR20	258.7	94.300 – 718.258
ELR50	368.9	78.395 – 1591.201
LOELR	1000.0	-
NOELR	316.0	-

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

Therefore, after 21 days, the NOELR value determined for the most sensitive endpoint cumulative number of offspring was 316 µg/L. The NOELR was observed at levels far above the water solubility of the test item and is not interpreted as pelagic toxicity.

Description of key information

The effects of a chronic exposure to test item TERPENIC OLIGOMERS on the reproduction of Daphnia magna was investigated. The NOELR value determined for the most sensitive endpoint cumulative number of offspring was 316 µg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

316 µg/L

Additional information

A study was performed to assess the effects of a chronic exposure to test item TERPENIC OLIGOMERS on the reproduction of freshwater crustaceanDaphnia magna. The followed method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 211, "Daphnia magna, Reproduction Test", referenced as Method C.20 of Commission Regulation No. 440/2008 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Daphnids were exposed toWater Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of10, 32, 100, 316 and 1000 µg/L,and to an untreated control and a solvent control for a total period of 21 days in closed vessels. Each treatment group consisted of 10 replicates each of oneDaphnia magna(one daphnia per replicate). The test media was renewed three times a week and the daphnidswere fed five days a week. Effects on reproductive performance were investigated by counting the living offspring produced by each parent animal every day. Mortality among the parent animals was also recorded. At the end of the test the length of the adults was measured by microscopy. The concentrations of the test item were determined by chemical analysesthree times a week at renewal of the test media.

Due to the very low solubility of the test item in water, it was not possible to quantify and detect the presence of the test item in the lowest loading rates, despite the use of acetone for the preparation of WAFs. Nevertheless, the test item was detected in the highest loading rates, and even quantified (> LOQ) at 316 and 1000 µg/L, but since control solutions were outside the tolerance limits those values cannot be considered as valid.Giventhe complex nature of the WAF and since the test item was a UVCB substance, the results were based on nominalloading rates.

This result is used as key value for chemical safety assessment.