Gum turpentine oil, consisting of the volatile fraction resulting from the distillation of oleoresin collected by tapping softwoods from pinacea derivatives (genus: pinus). It consists of terpenes, mainly monoterpenes such as alpha-pinene and beta-pinene.

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2018-01-18 to 2018-01-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 2017-01-10

Test material

Test material information

Constituent 1

Reference substance name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene; 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene; 3-methylidene-6-(propan-2-yl)cyclohex-1-ene; 6,6-dimethyl-2-methylidenebicyclo[3.1.1]heptane; 7-methyl-3-methylideneocta-1,6-diene

EC Number:

932-349-8

Cas Number:

8006-64-2

Molecular formula:

C10H16

IUPAC Name:

2,6,6-trimethylbicyclo[3.1.1]hept-2-ene; 3,7,7-trimethylbicyclo[4.1.0]hept-3-ene; 3-methylidene-6-(propan-2-yl)cyclohex-1-ene; 6,6-dimethyl-2-methylidenebicyclo[3.1.1]heptane; 7-methyl-3-methylideneocta-1,6-diene

Test material form:

liquid

Details on test material:

Name of test material (as cited in study report): Turpentine ; turpentine oil

Specific details on test material used for the study:

Description: Colourless – slightly yellow liquid
Batch No.: FAB-08082017
Test substance storage: Refrigeration (+2°C to +8°C) under nitrogen. Keep away from light and humidity.
Date of manufacture: 8 August 2017
Date of expiration: 7 August 2018

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Chemical analysis: Single samples for analysis were taken from the control and from all test concentrations according to the schedule in "any other information section". Samples for fresh solutions were taken from each treatment prior to addition of the daphnids and the suspension of algal cells.
Measurements and recordings:
- Offspring: The living offspring produced by each parent animal were removed and counted daily 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 (i.e. body length excluding the anal spine) was measured by microscopy using an ocular micrometer calibrated with a stage micrometer.

Test solutions

Vehicle:

no

Details on test solutions:

The study was carried out using a WAF (Water Accommodated Fraction), which was thereafter diluted to obtain the required test solutions, according to the OECD Guidance Document No. 23. The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the WAF. The volume of the mixing vessel was approximately 5 L. A magnetic stirring bar was placed in the vessel and 5.3 L to 5.9 L of test water (depending on the brim capacity of the bottle) were added in order to use a maximum volume and to minimise headspace. The loading rate of the test item was weighed on a glass slide that afterwards was placed under the surface of the test water contained in the mixing vessel through fishing wire, in order to obtain a stock solution at the nominal concentration of 2 mg.L-1. Then the mixing vessel was 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. The stirring speed was as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase. After 24 +/- 2 hours of gentle stirring (except for the renewal on the first Monday of the test where stirring was carried out for approx. 2 days) in the dark at room temperature, the WAF was extracted via the drain port. The first 100 mL were removed and then the WAF was diluted with test water as necessary to obtain the required test concentrations into 1-L volumetric flasks. Each flask was inverted several times before filling the test vessels (without headspace) to ensure adequate mixing and homogeneity of test solutions. 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. The test was carried out without adjustment of the pH.
At each renewal, the entire procedure was repeated when preparing fresh test solutions.

Tests concentrations:
- Test item: Based on results of acute toxicity tests on D. magna with the two main constituents (LPL Project D12-006 (alpha-pinene): 48h-EC50 = 0.475 mg.L-1 based on measured concentrations; LPL Project D14-004 (beta-pinene): 48h-EC50 = 1.25 mg.L-1 based on measured concentrations), test solutions used in the definitive test were prepared by direct addition of the required amounts of the stock solution (WAF) to test water to obtain the following nominal concentrations (spaced by a factor of 2.34): 10, 20, 50, 130 and 300 µg.L-1.
- Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms

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.
- Introduction of daphnids: Daphnids were introduced into the test medium immediately after filling the test bottles with test solutions and algal cells. At each renewal, daphnids were transferred from the old vessels to the freshly prepared test media.
- Feeding: Daphnids were fed every day with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1.

Study design

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 211

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Test conditions

Hardness:

140 mg CaCO3

Test temperature:

situated between 20.7 and 21.1°C (average: 20.9°C)

pH:

6.0-9.0, not varying by more than 1.5 units

Dissolved oxygen:

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

Salinity:

Not specified.

Conductivity:

Not specified.

Nominal and measured concentrations:

Test solutions used in the definitive test were prepared by direct addition of the required amounts of the stock solution (WAF) to test water to obtain the following nominal concentrations (spaced by a factor of 2.34): 10, 20, 50, 130 and 300 µg.L-1.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60-mL glass flasks sealed with assembled screw cap with hole and PTFE/silicone septum. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
- Type: closed conditions
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: daily renewal (except the week-end)
- Number of animals: 10 animals individually held at each test concentration and the control series
- Loading: 1 daphnid per test vessel each completely filled with test solution and with no headspace
- No. of vessels per concentration: 10
- No. of vessels per control (replicates): 10

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: test water: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 211. The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3).
- Intervals of water quality measurement:
- Dissolved O2, hardness, pH: Measured at least 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.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16h light : 8h dark
- Light intensity: not exceeding 1000 - 1500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Offspring, Mortality, Individual length of adults

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.34

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 300 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks:

living offspring

Details on results:

Mortalities of parents from some replicates were allocated as inadvertent mortalities (not treatment-related mortalities) and were thus excluded from the analysis in accordance with test guideline. Thus, the effect on reproduction was determined as the total number of living offspring produced per survived (= introduced) parent animal which did not die accidentally or inadvertently during the test, in agreement with the Study Monitor and OECD Guideline 211. As mentioned above, the evaluation of effects was based on nominal test concentrations.
No ECx could be determined due to the lack of effects at the tested concentrations. However, a significant percentage reduction of cumulative offspring was found only at 50 µg.L-1, which was not the case at higher test concentrations.
Regarding the analytical results, where concentrations in alpha- and beta-pinene were significantly much higher at 130 and 300 µg.L-1 than the ones at 50 µg.L-1, it was assumed that the percentage reduction of cumulative offspring observed at 50 µg.L-1 could not be related to an effect of the test item, in agreement with the Study Monitor. Therefore, the NOEC value for the parameter cumulative number of offspring was determined to be ≥ 300 µg.L-1. The NOEC value determined for length was < 10 µg.L-1.

Results with reference substance (positive control):

Not applicable.

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.

Any other information on results incl. tables

Detailed biological results

Reduction of cumulative offspring per survived (= introduced) parent caused by the test item after 21 d

 

Nominal concentration(µg.L^-1)	Mean	Std. Dev.	n	% Reduction
Control	84.1	9.16	9	-
10	84.0	8.60	10	0.1
20	80.0	12.06	8	4.9
50	69.4	8.90	10	17.5
130	80.7	10.40	9	4.1
300	83.1	9.98	9	1.2

Std. Dev.: standard deviation; n: number of replicates.

Values were excerpted from the computer program ToxRat.

No. Live offspring per replicate and day.

 

Controls	Day
Replicate	8	9	10	11	12	13	14	15	16	17	18	19	20	21	Total
1	10	0	0	17	0	0	22	0	0	5	18	0	0	19	91
2	0	9	0	0	14	0	2	19	0	0	22	0	0	18	84
3	0	16	0	0	17	0	0	3	16	0	0	17	0	0	69
4	0	0	16	0	4	18	0	1	22	0	0	23	0	0	84
5	0	15	0	0	15	0	0	1	17	0	M	M	M	M	-
6	0	11	0	0	17	1	0	20	0	0	16	1	0	15	81
7	0	10	0	0	16	0	0	20	0	0	18	0	0	15	79
8	0	14	0	0	18	0	0	14	0	0	17	0	0	20	83
9	3	7	0	12	5	0	8	12	0	5	15	0	0	16	83
10	0	13	0	0	20	0	2	23	0	0	24	0	0	21	103
Cumulative mortality of parental animals	-	-	-	-	-	-	-	-	-	-	1	1	1	1	1
Mean Offspring Number (Cumulative offspring per survived/introduced parent):84.1

10 µg.L^-1	Day
Replicate	8	9	10	11	12	13	14	15	16	17	18	19	20	21	Total
1	0	13	0	0	21	0	0	5	15	0	0	19	0	0	73
2	0	13	0	0	21	0	0	3	17	0	0	23	0	0	77
3	0	15	0	0	19	0	0	0	22	0	0	24	0	0	80
4	0	8	1	0	15	0	0	20	0	0	20	0	0	14	78
5	0	11	0	0	16	0	0	21	0	0	21	0	0	14	83
6	10	0	0	19	0	0	22	0	0	16	7	0	0	21	95
7	11	0	0	18	0	0	23	0	0	23	0	0	0	22	97
8	10	0	0	20	0	0	23	0	0	24	0	0	2	15	94
9	0	15	0	0	20	1	0	28	0	0	0	22	0	0	86
10	0	14	0	0	21	0	0	9	12	0	5	16	0	0	77
Cumulative mortality of parental animals	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Mean Offspring Number (Cumulative offspring per survived/introduced parent):84.0

 “M”: dead females

 

20 µg.L^-1	Day
Replicate	8	9	10	11	12	13	14	15	16	17	18	19	20	21	Total
1	0	15	0	0	14	1	0	2	22	0	0	21	0	0	75
2*	M	M	M	M	M	M	M	M	M	M	M	M	M	M	-
3	0	0	11	0	0	13	M	M	M	M	M	M	M	M	-
4	0	13	0	0	24	0	0	21	0	0	4	16	0	5	83
5	8	1	0	20	0	0	18	0	0	3	15	0	0	18	83
6	0	11	0	0	19	0	0	0	0	0	5	20	0	0	55
7	0	15	0	0	19	0	0	5	17	0	1	23	0	0	80
8	0	14	0	0	16	0	0	20	0	0	26	0	0	22	98
9	0	11	4	0	21	0	0	3	21	0	0	21	0	0	81
10	10	0	0	17	0	0	22	0	0	20	0	0	16	0	85
Cumulative mortality of parental animals	1	1	1	1	1	1	2	2	2	2	2	2	2	2	2
Mean Offspring Number (Cumulative offspring per survived/introduced parent):80.0

“M”: dead females

*This replicate was found dead since day 5.

Replicates 2 and 3 excluded from calculations (mortality considered as not related to the test substance

50 µg.L^-1	Day
Replicate	8	9	10	11	12	13	14	15	16	17	18	19	20	21	Total
1	0	5	0	0	11	0	0	12	0	0	16	0	0	14	58
2	0	5	0	0	11	0	0	14	0	0	12	0	0	14	56
3	0	1	0	0	9	0	0	16	0	6	14	0	0	15	61
4	0	4	0	0	10	0	0	17	0	0	17	0	0	23	71
5	0	5	0	0	13	0	0	15	0	0	18	0	0	19	70
6	0	13	0	0	21	0	0	12	0	0	17	4	0	0	67
7	0	15	0	0	21	0	0	4	18	0	0	24	0	0	82
8	0	10	0	0	17	0	0	10	0	0	19	0	0	19	75
9	0	11	0	0	7	0	0	15	0	0	21	0	0	20	74
10	10	1	0	16	2	0	0	11	0	3	17	0	0	20	80
Cumulative mortality of parental animals	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Mean Offspring Number (Cumulative offspring per survived/introduced parent):69.4

  “M”: dead females

 

130 µg.L^-1	Day
Replicate	8	9	10	11	12	13	14	15	16	17	18	19	20	21	Total
1	9	0	0	17	0	0	24	0	0	17	2	0	0	18	87
2	0	7	0	1	13	0	0	19	0	0	18	0	0	19	77
3	0	11	0	0	18	0	0	14	11	0	1	22	0	0	77
4	12	0	0	23	0	0	23	0	0	11	10	0	0	19	98
5*	M	M	M	M	M	M	M	M	M	M	M	M	M	M	-
6	0	2	13	0	0	20	0	0	23	0	0	22	0	0	80
7	0	0	12	0	0	16	0	0	18	0	0	19	0	0	65
8	0	13	0	0	15	0	0	0	20	0	0	23	0	0	71
9	0	16	0	0	20	0	0	20	0	0	4	17	0	1	78
10	11	0	0	17	0	0	24	0	0	6	18	0	0	17	93
Cumulative mortality of parental animals	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
Mean Offspring Number (Cumulative offspring per survived/introduced parent):80.7

“M”: dead females

*This replicate was found dead since day 5.

Replicate 5 excluded from calculations (mortality considered as not related to the test substance).

 

 

300 µg.L^-1	Day
Replicate	8	9	10	11	12	13	14	15	16	17	18	19	20	21	Total
1	12	0	0	16	0	0	23	0	0	3	20	0	0	20	94
2	0	4	13	0	2	16	0	0	23	0	0	20	0	0	78
3	0	10	0	0	16	0	0	19	0	0	15	0	0	4	64
4	9	0	0	14	0	0	21	1	0	4	14	0	0	18	81
5*	M	M	M	M	M	M	M	M	M	M	M	M	M	M	-
6	3	8	0	11	3	0	18	5	0	4	16	0	0	19	87
7	6	6	0	18	0	0	21	0	0	0	24	0	0	20	95
8	11	0	0	15	0	0	23	0	0	16	2	0	4	15	86
9	0	13	0	0	18	0	0	20	0	0	10	8	0	5	74
10	13	1	0	14	0	0	24	0	0	3	16	0	0	18	89
Cumulative mortality of parental animals	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1
Mean Offspring Number (Cumulative offspring per survived/introduced parent):83.1

 

“M”: dead females

*This replicate was found dead since day 5.

Replicate 5 excluded from calculations (mortality considered as not related to the test substance).

 

Cumulative mortality of parental animals during the test

	Nominal concentration(µg.L^-1)
Day	Control	10	20	50	130	300
1	0	0	0	0	0	0
2	0	0	0	0	0	0
3	0	0	0	0	0	0
4	0	0	0	0	0	0
5	0	0	0 (1)	0	0 (1)	0 (1)
6	0	0	0	0	0	0
7	0	0	0	0	0	0
8	0	0	0	0	0	0
9	0	0	0	0	0	0
10	0	0	0	0	0	0
11	0	0	0	0	0	0
12	0	0	0	0	0	0
13	0	0	0	0	0	0
14	0	0	0 (1)	0	0	0
15	0	0	0	0	0	0
16	0	0	0	0	0	0
17	0	0	0	0	0	0
18	0 (1)	0	0	0	0	0
19	0	0	0	0	0	0
20	0	0	0	0	0	0
21	0	0	0	0	0	0
Mortality %	0	0	0	0	0	0

(1) Mortality not related to treatment (inadvertent mortality), replicates excluded. 

 

Cumulative offspring per introduced/survived parent at the end of the test.

 

Replicate	Nominal concentration(µg.L^-1)
	Control	10	20	50	130	300
1	91	73	75	58	87	94
2	84	77	-*	56	77	78
3	69	80	-*	61	77	64
4	84	78	83	71	98	81
5	-*	83	83	70	-*	-*
6	81	95	55	67	80	87
7	79	97	80	82	65	95
8	83	94	98	75	71	86
9	83	86	81	74	78	74
10	103	77	85	80	93	89
Mean	84.1	84.0	80.0	69.4	80.7	83.1
Std. Dev	9.16	8.60	12.06	8.90	10.40	9.98
n	9	10	8	10	9	9
CV	10.9	10.2	15.1	12.8	12.9	12.0

*Replicates excluded from calculations (inadvertent mortality, considered as not related to the test item).

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

Parental Daphnia magna lengths after 21 days (mm).

 

 

Replicate	Nominal concentration(µg.L^-1)
	Control	10	20	50	130	300
1	3.65	3.75	3.60	3.40	3.45	3.60
2	3.75	3.60	M	3.30	3.50	3.50
3	3.50	3.40	M	3.30	3.30	3.45
4	3.75	3.50	3.70	3.50	3.60	3.75
5	M	3.60	3.65	3.55	M	M
6	3.60	3.80	3.25	3.65	3.70	3.50
7	3.75	3.70	3.50	3.20	3.40	3.50
8	3.70	3.70	3.80	3.60	3.60	3.50
9	3.80	3.50	3.55	3.60	3.50	3.50
10	3.90	3.25	3.65	3.50	3.60	3.80
Mean	3.71	3.58	3.59	3.46	3.52	3.57
Std. Dev	0.117	0.170	0.164	0.152	0.122	0.125
n	9	10	8	10	9	9
CV	3.1	4.8	4.6	4.4	3.5	3.5

“M”: dead females (excluded from the calculations)

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

 

 

Analytical results

Chemical analyses were performedon the mainconstituents(alpha-pinene and beta-pinene)to provide an indication of the stability of test solutions prepared from the WAF at 2 mg.L^-1.

Although every effort was made to maintain the concentrations of the test item (i.e.daily renewal of test solutionsand closed conditions without headspace), the loss of test item displayed by the quantification of the key elements of the test item was sometimes greater than 20% of the measured initial concentration. In other words, test item levels were overall stable, especially at the highest test concentration (300 µg.L^-1, nominal), where the main constituents were generally close to or within the 20% of the initial concentration. However, actual levels of each constituent were lower than the expected ones. It should be noted that test solutions were prepared from a WAF, and that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the same way that if it was alone in the mixture.

Based on these results some difference between the nominal and measured concentrations values was found as expected, but the main constituents were overall maintained. Therefore, and since the test item was a UVCB substance,the evaluation of the effects was based on nominal test concentrations, in agreement with the Study Monitor.

Concentrations (µg.L^-1) of the test item in test water – Final test – Alpha-pinene (75.0% test item)

 

		Nominal concentration(µg test item.L^-1) and expected concentrations in Alpha-pinene
		Control	10.00	20.00	50.00	130.00	300.00
Days		0	7.50	15.00	37.50	97.50	225.00
0 - 3	Fresh	Presence*	4.11	8.41	14.66	35.55	95.33
	Old	Absence	2.12	4.68	13.30	43.66	98.79
	Relative loss to initial value (%)	N.A.	48.28	44.32	9.30	-22.81	-3.63
4 - 5	Fresh	Absence	2.01	3.28	9.42	29.19	77.15
	Old	Absence	Presence	3.00	10.34	21.13	67.06
	Relative loss to initial value (%)	N.A.	N.A.	8.63	-9.69	27.60	13.07
6 - 7	Fresh	Absence	Presence	3.48	12.17	18.90	51.81
	Old	Absence	1.35	3.47	5.95	13.85	41.62
	Relative loss to initial value (%)	N.A.	N.A.	0.37	51.12	26.70	19.68
10 -11	Fresh	Absence	3.02	4.55	8.67	33.87	71.80
	Old	Absence	2.04	3.40	9.38	17.28	53.81
	Relative loss to initial value (%)	N.A.	32.24	25.18	-8.14	49.00	25.06
11 - 12	Fresh	Absence	3.98	2.26	13.54	28.75	85.35
	Old	Absence	2.24	4.24	13.44	22.83	58.43
	Relative loss to initial value (%)	N.A.	43.63	-87.54	0.69	20.58	31.54
12 - 13	Fresh	Absence	3.76	3.13	9.98	32.77	64.00
	Old	Absence	2.76	2.53	9.61	20.57	53.87
	Relative loss to initial value (%)	N.A.	26.81	19.20	3.72	37.21	15.84
17 - 18	Fresh	Absence	Presence	2.56	5.12	11.22	27.91
	Old	Absence	1.80	2.27	5.66	13.61	35.26
	Relative loss to initial value (%)	N.A.	N.A.	1.35	-10.53	-21.26	-26.32
19 - 20	Fresh	Absence	4.44	7.52	14.73	46.98	122.98
	Old	Absence	3.34	7.19	15.34	29.01	93.63
	Relative loss to initial value (%)	N.A.	24.81	4.30	-4.17	38.25	23.86
20 - 21	Fresh	Absence	2.29	9.45	18.66	35.50	92.27
	Old	Absence	3.01	10.16	16.89	31.47	74.71
	Relative loss to initial value (%)	N.A.	-31.50	-7.55	9.49	11.35	19.03

N.A.: not applicable

Presence: concentrations below the LOQ (1.16 µg.L^-1) but above the LOD (0.35 µg.L^-1).

Absence: concentrations below the LOQ (1.16 µg.L^-1) and the LOD (0.35 µg.L^-1).

*A slight contamination was observed in the control, but it was just over the LOD (trace of the test item). This contamination was considered not to affect the results of the test as no adverse effects of exposure were observed in the control throughout the duration of the test.

 

 

 Concentrations (µg.L^-1) of the test item in test water – Final test – Beta-pinene (16.2% test item)

 

		Nominal concentration(µg test item.L^-1) and expected concentrations in Beta-pinene
		Control	10.00	20.00	50.00	130.00	300.00
Days		0	1.62	3.24	8.10	21.06	48.60
0 - 3	Fresh	Presence*	1.62	2.93	4.85	12.00	31.48
	Old	Absence	Presence	1.89	4.36	13.46	30.50
	Relative loss to initial value (%)	N.A.	N.A.	35.55	9.99	-12.11	3.11
4 - 5	Fresh	Presence*	Presence	1.44	3.35	9.63	24.49
	Old	Presence*	Presence	1.38	3.73	7.42	22.54
	Relative loss to initial value (%)	N.A.	N.A.	4.65	-11.50	22.90	7.95
6 - 7	Fresh	Presence*	Presence	1.76	4.71	7.42	19.61
	Old	Absence	Presence	1.69	2.62	5.25	14.86
	Relative loss to initial value (%)	N.A.	N.A.	3.98	44.33	29.21	24.25
10 -11	Fresh	Absence	Presence	1.56	3.04	11.13	23.41
	Old	Absence	Presence	1.30	3.40	6.13	18.65
	Relative loss to initial value (%)	N.A.	N.A.	16.85	-11.86	44.88	20.34
11 - 12	Fresh	Absence	1.24	Presence	3.91	8.71	25.71
	Old	Absence	Presence	1.37	3.87	6.34	17.58
	Relative loss to initial value (%)	N.A.	N.A.	N.A.	1.05	27.20	31.65
12 - 13	Fresh	Absence	1.40	Presence	3.34	11.34	20.78
	Old	Absence	Presence	Presence	3.08	6.70	17.72
	Relative loss to initial value (%)	N.A.	N.A.	N.A.	7.75	40.90	14.75
17 - 18	Fresh	Absence	Presence	1.36	2.50	6.04	13.71
	Old	inv. data	inv. data	inv. data	inv. data	inv. data	inv. data
	Relative loss to initial value (%)	N.A.	N.A.	N.A	N.A	N.A	N.A
19 - 20	Fresh	Absence	1.50	2.38	4.61	13.66	35.94
	Old	Absence	1.25	2.25	4.53	8.31	27.60
	Relative loss to initial value (%)	N.A.	16.58	5.47	1.80	39.18	23.20
20 - 21	Fresh	Absence	Presence	2.85	5.39	10.36	26.31
	Old	Absence	Presence	3.05	4.75	9.61	19.96
	Relative loss to initial value (%)	N.A.	N.A.	-7.31	11.90	7.27	24.14

N.A.: not applicable

inv. data: invalidated data (control solutions outside the acceptable range).

Presence: concentrations below the LOQ (1.16 µg.L^-1) but above the LOD (0.35 µg.L^-1).

Absence: concentrations below the LOQ (1.16 µg.L^-1) and the LOD (0.35 µg.L^-1).

*A slight contamination was observed in the control, but it was just over the LOD (trace of the test item). This contamination was considered not to affect the results of the test as no adverse effects of exposure were observed in the control throughout the duration of the test.

pH-values throughout the test

Day		Nominal concentration(µg.L^-1)
		Control	10	20	50	130	300
0	Fresh	8.27	8.29	8.30	8.30	8.28	8.34
3	Old	8.57	8.50	8.51	8.48	8.40	8.51
	Fresh	8.18	8.37	8.41	8.27	8.38	8.29
4	Old	8.10	8.22	8.13	8.26	8.25	8.26
	Fresh	8.03	8.20	8.26	8.24	8.23	7.98
5	Old	7.73	8.03	8.04	8.16	8.10	8.00
	Fresh	8.24	8.22	8.22	8.30	8.28	8.20
6	Old	7.56	7.34	7.92	8.12	7.94	7.95
	Fresh	8.71	7.94	8.20	8.36	8.22	8.26
7	Old	8.05	8.00	8.14	8.10	8.08	7.98
	Fresh	8.23	8.20	8.21	8.25	8.23	8.29
8	Old	8.01	8.07	8.03	8.03	7.90	7.99
	Fresh	8.31	8.23	8.26	8.29	8.18	8.12
9	Old	7.68	8.10	7.98	8.10	8.01	8.01
	Fresh	8.26	8.31	8.21	8.32	8.16	8.10
10	Old	7.79	7.80	7.94	8.11	7.87	7.91
	Fresh	8.07	8.26	8.27	8.21	8.20	8.20
11	Old	7.70	7.90	8.07	7.96	7.99	7.94
	Fresh	8.17	8.25	8.23	8.20	8.18	8.12
12	Old	7.75	7.66	7.71	7.86	7.79	7.99
	Fresh	8.20	8.28	8.22	8.26	8.15	8.18
13	Old	7.86	8.08	7.74	7.76	7.81	7.78
	Fresh	8.51	8.59	8.59	8.59	8.54	8.46
14	Old	7.72	7.96	8.40	8.19	7.80	8.02
	Fresh	8.53	8.52	8.58	8.56	8.46	8.55
15	Old	7.80	8.01	8.36	7.80	7.79	7.90
	Fresh	8.50	8.46	8.47	8.45	8.27	8.50
16	Old	7.71	8.04	8.10	7.89	7.93	7.79
	Fresh	7.75	8.43	8.38	8.43	8.41	8.43
17	Old	7.72	8.03	7.76	7.92	7.81	7.78
	Fresh	8.93	8.27	8.42	8.37	8.50	8.41
18	Old	7.78	7.94	8.03	7.98	7.96	7.82
	Fresh	8.07	8.25	8.23	8.27	8.19	8.29
19	Old	7.64	7.96	8.01	8.12	7.67	7.74
	Fresh	8.16	8.33	8.26	8.39	8.29	8.38
20	Old	7.76	7.86	8.07	7.95	8.10	7.91
	Fresh	8.33	8.30	8.36	8.40	8.40	8.37
21	Old	7.84	8.15	8.15	8.07	7.97	8.15

Dissolved oxygen values (mg.L^-1) throughout the test

 

Day		Nominal concentration(µg.L^-1)
		Control	10	20	50	130	300
0	Fresh	8.57	8.56	8.42	8.50	8.53	8.47
3	Old	8.93	8.98	9.09	8.90	8.80	9.25
	Fresh	8.73	8.76	8.70	8.69	8.68	8.66
4	Old	8.58	8.57	8.60	8.70	8.61	8.60
	Fresh	8.62	8.60	8.65	8.62	8.55	8.65
5	Old	8.48	8.33	8.44	8.55	8.47	8.41
	Fresh	8.82	8.76	8.74	8.84	8.79	8.58
6	Old	8.49	8.28	8.47	8.60	8.35	8.30
	Fresh	8.58	8.52	8.52	8.50	8.51	8.47
7	Old	8.01	7.87	7.85	7.93	7.95	7.85
	Fresh	8.52	8.45	8.42	8.46	8.41	8.36
8	Old	8.12	8.01	7.99	7.99	8.01	8.01
	Fresh	8.51	8.51	8.51	8.54	8.57	8.41
9	Old	8.01	7.89	7.87	7.90	7.90	7.88
	Fresh	8.61	8.26	8.46	8.43	8.43	8.48
10	Old	8.17	8.22	8.09	8.29	7.98	8.01
	Fresh	8.74	8.62	8.56	8.53	8.50	8.53
11	Old	8.01	7.72	7.82	8.13	7.57	8.03
	Fresh	8.70	8.73	8.65	8.66	8.66	8.46
12	Old	7.01	7.60	7.42	7.96	7.53	8.01
	Fresh	8.90	8.92	8.92	8.87	8.89	8.83
13	Old	7.20	7.44	7.27	7.50	7.19	7.14
	Fresh	9.08	9.03	9.09	9.03	9.02	9.01
14	Old	7.47	7.60	8.76	7.76	7.61	7.66
	Fresh	9.30	8.99	9.17	9.10	9.08	9.07
15	Old	7.04	7.40	8.70	7.51	7.80	7.80
	Fresh	9.01	9.02	9.04	8.96	8.69	8.89
16	Old	7.59	7.60	8.10	7.62	7.58	7.34
	Fresh	8.93	8.70	8.92	8.51	8.52	8.52
17	Old	8.02	8.01	8.86	7.56	7.70	7.72
	Fresh	8.97	8.90	9.07	9.07	8.99	8.90
18	Old	8.04	8.07	7.96	7.89	7.70	7.70
	Fresh	8.46	8.41	8.31	8.25	8.22	8.43
19	Old	8.12	8.47	8.36	8.40	7.70	7.88
	Fresh	9.80	9.80	9.80	9.98	9.87	9.91
20	Old	7.60	7.57	8.20	7.38	8.17	7.98
	Fresh	8.93	8.89	8.97	9.01	8.85	8.82
21	Old	8.17	8.80	8.57	8.38	8.21	8.48

Water temperature (°C) throughout the test 

Day		Nominal concentration(µg.L^-1)
		Control	10	20	50	130	300
0	Fresh	20.3	19.9	19.9	19.8	19.7	19.8
3	Old	20.5	20.6	21.3	20.6	20.5	20.4
	Fresh	21.6	21.4	21.5	21.2	21.4	21.5
4	Old	20.5	20.5	20.1	20.7	20.7	20.1
	Fresh	20.4	20.4	20.5	20.5	20.3	20.5
5	Old	20.4	20.5	20.5	20.5	20.6	20.3
	Fresh	20.3	20.3	20.4	20.4	20.3	20.5
6	Old	20.7	20.1	20.3	20.3	20.2	20.3
	Fresh	20.3	20.3	20.1	20.4	20.4	20.3
7	Old	20.3	20.7	20.3	20.7	20.7	20.7
	Fresh	20.8	20.5	20.5	20.8	20.5	20.4
8	Old	20.9	20.7	20.6	20.7	20.7	20.9
	Fresh	20.8	20.9	20.7	20.9	20.7	20.7
9	Old	20.3	20.4	20.4	20.5	20.5	20.4
	Fresh	20.8	20.8	20.8	20.8	20.8	20.8
10	Old	20.8	20.8	20.6	20.7	20.6	20.7
	Fresh	20.4	20.7	20.8	20.7	20.8	20.8
11	Old	20.6	20.7	20.3	20.5	20.6	20.6
	Fresh	20.7	20.8	20.7	20.7	20.7	20.8
12	Old	21.4	20.6	20.6	20.6	20.7	20.6
	Fresh	20.5	19.8	20.4	20.4	20.6	20.7
13	Old	21.1	21.0	20.9	21.0	20.9	20.8
	Fresh	20.6	20.4	20.6	20.7	20.7	20.6
14	Old	20.9	20.6	20.7	20.8	20.9	20.9
	Fresh	20.4	20.6	20.6	20.6	20.6	20.6
15	Old	20.8	20.8	20.8	20.9	20.8	20.8
	Fresh	20.6	20.7	20.6	20.6	20.6	20.6
16	Old	20.9	20.8	20.8	20.9	20.8	20.9
	Fresh	21.1	21.1	21.1	21.1	20.9	21.1
17	Old	20.9	20.9	20.9	20.9	20.9	20.8
	Fresh	21.0	21.0	20.8	20.8	20.9	21.0
18	Old	21.1	20.8	21.0	20.7	20.7	20.9
	Fresh	20.9	21.0	20.9	20.9	20.9	20.8
19	Old	21.0	20.9	20.9	20.8	20.8	20.8
	Fresh	20.9	20.8	20.9	20.9	20.9	20.9
20	Old	20.9	20.9	20.8	20.7	20.7	20.7
	Fresh	20.8	20.7	20.7	20.8	20.8	20.9
21	Old	20.8	20.7	20.6	20.7	20.8	20.8

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effects of a chronic exposure to test item GUM TURPENTINE OIL on the reproduction of Daphnia magna was investigated in this study. The evaluation was based on nominal test concentrations. Under the experimental conditions and after 21 days, the NOEC value for the most sensitive parameter cumulative number of offspring was determined to be at least (or higher than) 300 µg.L-1.

Executive summary:

A study was performed to assess the effects of a chronic exposure to test item GUM TURPENTINE OIL on the reproduction of the 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).

 

Based on results of acute toxicity tests on D. magna with the two main constituents (LPL Project D12-006 (alpha-pinene): 48h-EC₅₀= 0.475 mg.L^-1based on measured concentrations; LPL Project D14-004 (beta-pinene): 48h-EC₅₀= 1.25 mg.L^-1based on measured concentrations), daphnids were exposed to aqueous solutions of the test itemat the required nominal test concentrations10, 20, 50, 130 and 300 µg.L^-1,and to an untreated 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 every day,except the first weekend of the test, and the daphnidswere fed at each renewal. 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, represented by the quantification of the two main constituents, were determined by chemical analyses three times a week.

 

Test item levels were overall stable, especially at the highest test concentration (300 µg.L^-1, nominal), where the main constituents were generally close to or within the 20% of the initial concentration. However, actual levels of each constituent were lower than the expected ones.Since the test item was a UVCB substance, the evaluation of the effects was based on nominal test concentrations. No ECx could be determined due to the lack of effects at the tested concentrations. However, a significant percentage reduction of cumulative offspring (17.5%) was found only at 50 µg.L^-1, but not at higher test concentrations. Regarding the analytical results, where concentrations in alpha- and beta-pinene were significantly much higher at 130 and 300 µg.L^-1 than the ones at 50 µg.L^-1, it  was assumed that the percentage reduction of cumulative offspring observed at 50 µg.L^-1could not be related to an effect of the test item, in agreement with the Study Monitor. Therefore, the NOEC value forthe parameter cumulative number of offspring was determined to be ≥ 300 µg.L^-1.