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Long-term toxicity to aquatic invertebrates

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Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 January to 18 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline No. 211. All validity criteria were fulfilled.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
PHYSICO-CHEMICAL PROPERTIES
- Density (20 °C): 0.842 g/mL
- Water solubility (20 °C): 4.0-5.7 mg/L
- Vapour pressure (298 °K): 200 Pa
Analytical monitoring:
yes
Details on sampling:
In order to verify the test concentrations, duplicate samples were collected from the additional test flasks without daphnids and algae for each test concentration and the control at the initiation of the test (0 h), at each renewal (old and new test solutions) and at the termination of the test. Duplicate samples of the stock solutions B prepared for each renewal were also collected. 10 ±1 mL of each sample were collected in 20-mL headspace vials sealed with aluminium crimp caps and stored at -20 ± 2.0 °C. The headspace vials were used directly in the autosampler at the analytical laboratory LPL. Although duplicate samples were collected of each test concentrations and stock solution B, only one of these samples was analysed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A saturated stock solution (A) of d-Limonene was prepared by using the following procedure:
A 2-liter glass bottle with a magnet (total content 2.22 L) was filled with 2.19 L medium. A pipette tip was saturated by filling it 3 times with the test item. 7 mL of the test item was added slowly to the 2-liter glass bottle to form a thin layer of approximately 5 mm thickness. The bottle was rapidly sealed tightly with a screw cap with PTFE packing. The bottle was covered with black cloth and the stirring was turned on in order to observe a vortex of about 2 cm (approximately 275 rpm), at room temperature for 48 ± 2 h. The stirring was stopped and after 1 h, approximately 1100 mL of the mid fraction was syphoned off using double tube connected to a glass tube with narrow diameter. The first 100 mL of the mid fraction was discarded and the rest transferred to a new glass bottle with as little headspace as possible (stock solution B). When transferring the stock solution precautions were taken to minimise loss of the test item to the air by gently pouring solutions into the flask, below the surface of the solution when possible. The test solutions were prepared individually by immediately diluting stock solution B in test medium to achieve the following concentration series: 2.5, 4.0, 6.5, 10 and 16 % of the saturated solution.
At each renewal, the entire procedure was repeated when preparing fresh test solutions. The test solution were poured into clean test vessels. As the pH in all the stock solutions prepared during the test were 7.6-7.9, no pH adjustment was necessary.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Test species: Daphnia magna (Straus)
- Strain/clone: Not reported
- Source: Daphnids were collected in the Langedam, Birkerød, and cultured at test facility since 1979
- Feeding: Culture is fed with Pseudokirchneriella subcapitata (clone: NIVA, CHL 1) three times a day by a peristaltic pump system
- Culture is renewed once a week by discarding the oldest culture and preparing a new culture with one to four day old offspring taken from the oldest cultures. Offspring from these cultures were used in the study avoiding the first progeny.
- Feeding during test: The alga Pseudokirchneriella subcapitata was added to the test solutions as feed, approx. 2.2 x 10^7 cells ≈ 0.15 mg C/adult animal/day until day 8, after which 0.2 mg C/adult animal/day was applied. On day 17 and day 19 extra food (0.2 mg C/adult animal) was added in all test flasks due to the large number of offspring.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Post exposure observation period:
None
Hardness:
Water hardness was 267 mg CaCO3/L (preparation made on 15 December 2015) and 263 mg CaCO3/L (preparation made on 19 January 2016),
Test temperature:
19.9 ± 0.1 °C (nominal)
pH:
Min.-Max. measured value: 7.7-9.4
Dissolved oxygen:
Min.-Max. measured value: 98-100% ASV
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 2.5, 4.0, 6.5, 10 and 16 % of the saturated solution in the test medium (corresponding to TWM: 23, 50, 80, 173 and 363 μg/L, respectively)
Details on test conditions:
TEST SYSTEM
- Test vessel: Test was performed in 50-mL glass flasks sealed with PTFE-coated screw caps.
- Type: Closed; As the test item is volatile, a closed test system with a minor headspace was used.
- At the initiation of the test, the flasks were filled with test solution and 3 mL was withdrawn to have an equal headspace of 3 mL in each flask. Thereafter one female less than 24 h old was added to each test flask. The flasks were shaken gently on a shaking table during the whole test.
- Renewal rate of test solution: Each female was transferred to a new clean flask with a freshly prepared test solution every Monday, Wednesday and Friday. pH and oxygen saturation were recorded before and after each renewal of test solutions. Clean test flasks were used at every water renewal.
- Replicates: Ten daphnids were exposed to each test concentration.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7 medium was used in the test (according to OECD 211)

OTHER TEST CONDITIONS
- Photoperiod: Daily light/dark period of 16:8 h
- Light intensity: Climate room with normal laboratory light (approx. 8 μmol/(m2 x sec)

EFFECT PARAMETERS MEASURED
The following parameters were recorded at each renewal of test solutions: Number of offspring; mortality among parent animals; time at which the first offspring were present; aborted broods and dead offspring
- At the end of the test, the total number of live offspring produced per female alive at the end of the test was assessed. As the mortality amongst parent animals did not follow a concentration-response pattern, those replicates with parental mortality were excluded from the analysis of the test result. The reproductive output of the animals exposed to the test item was determined as the no observed effect concentration (NOEC) and the lowest effect concentration (LOEC). The 10 and 50 % effect concentrations (EC10 and EC50) were also determined.

RANGE-FINDING STUDY
- Based on the effects observed and analytical results in a pre-study (non-GLP) for the Daphnia magna reproduction test, the test item were tested at the following nominal concentrations: 0 (control), 2.5, 4.0, 6.5, 10 and 16 % of the saturated solution. The concentrations were chosen in such a way that the lowest concentration should cause no effect and the highest concentration should cause no parent mortality and 100 % inhibition of the reproduction, with the aim to establish an EC10 and EC50. An extra set of control replicates was included in open test beakers according to the normal procedure described in OECD 211, in order to investigate the difference between reproduction in the closed system used in this study and an open test system. 10 beakers with approx. 60 mL of test medium were included.
Reference substance (positive control):
no
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
153 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Number of live offspring produced per female alive at the end of the test
Remarks on result:
other: 95%CL: 83-222
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
188 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Number of live offspring produced per female alive at the end of the test
Remarks on result:
other: 95% CL: 118-257
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
80 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Number of live offspring produced per female alive at the end of the test
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
173 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
other: Number of live offspring produced per female alive at the end of the test
Details on results:
- The first offspring were observed after 8-10 days in the controls.
- Few aborted broods were observed whereas dead offspring was observed in all test concentrations, indicating that the test item is affecting the survival of the produced off-spring.
- Very few offspring was observed at the highest tested concentration.
Reported statistics and error estimates:
The statistical analyses were performed using the free software R (version 3.0.3 - 2014-03-06). A Probit analysis was conducted to estimate the EC10 and EC50. The LOEC/NOEC values were determined by use of the Dunnett’s test. The NOEC values (p>0.05) were determined as the highest tested concentration, at which no significant effect was observed compared with the control. The LOEC (t<0 and p<0.05) is the concentration just above the NOEC. To comply with the conditions of the Dunnett’s test, the normality of the data was assessed with a Shapiro-Wilk normality test. The variance was analysed with a Levene's test for homogeneity of variance.

Table 6.1.4/1: Endpoints observed in the 21-day reproduction test with Daphnia magna

 

Actual concentrations (TWM) μg/L

 

Average number of offspring

 

Mortality among parent animals (%)

 

Average time to which the first offspring were present (days)

 

Average number of dead offspring

 

Average number of aborted broods per replicate

0

63.6

 0

 8.7

 3.1

 1

23

80.1

 0

 8.9

 0.5

 0

50

88.4

 10

 9.1

 0.4

 0.3

80

76.4

 0

 9.0

 0.3

 0.1

173

42.8

 10

 9.7

 0

 0

363

1.5

 0

 > 21

 0

 0

 

pH raises in all test concentrations because the test is performed in closed flasks. Only in the highest test concentrations, pH are above 9, which is due to the low number of offspring available to eat the algae, which is causing the pH rise. The oxygen saturations recorded simultaneously is >95 % during the whole test in all test concentrations.

Validation                                

The validation criteria of the test guideline for the control series are fulfilled:

- The total male development and mortality of parent animals in the control did not exceed 20 % at the end of the test, as no females died and there were no males observed.

- The mean number of live offspring produced per female animal alive at the end of the test was ≥60. The result was 63.9 offspring per female.

Validity criteria fulfilled:
yes
Conclusions:
Taking into account the number of living offspring per surviving female as effect parameter, the EC10 and EC50 were 153 µg/L (95% CL 83-222 µg/L) and 188 µg/L (95% CL 118-257 µg/L), respectively. The NOEC was 80 µg/L and the LOEC was 173 µg/L. The effect parameters are based on the TWM of the actual measured concentration.
Executive summary:

A study was conducted to test the inhibitory effects of d-limonene on the reproduction of freshwater crustacean Daphnia magna according to Guideline OECD 211 and in compliance with GLP.

d-Limonene was tested at the following nominal concentrations: 0 (control), 2.5, 4, 6.5, 10 and 16% of a saturated solution of the test item in the test medium. As the test item is volatile, a closed test system with a minor headspace was used. The test was carried out as a semi-static test in 50-mL glass flasks sealed with PTFE-coated screw caps. All test solutions were renewed every Monday, Wednesday and Friday. The concentrations of the test item were determined by chemical analyses during the exposure period. The actual test concentrations of d-limonene was used in the calculation of the Time Weighted Mean (TWM), which was applied in the data analysis of the effect concentrations.

 

TWM of the measured concentrations were 23, 50, 80, 173 and 363 μg/L.

 

The first offspring was observed after 8-10 days in the controls. Few aborted broods were observed whereas dead offspring was observed in all test concentrations and in the controls, indicating that the effect was not treatment-related. Very few offspring was observed at the highest tested concentration. Mean number of live offspring produced per female animal alive at the end of the test were 63.9, 80.1, 88.4, 76.4, 42.8 and 1.5 offspring per female at 0, 23, 50, 80, 173 and 363 μg/L.

 

Therefore, taking into account the number of living offspring per surviving female as effect parameter, the EC10 and EC50 were 153 µg/L (95% CL: 83 -222 µg/L) and 188 µg/L (95%CL: 118 -257 µg/L), respectively. The NOEC was 80 µg/L and the LOEC was 173 μg/L. The effect parameters are based on the TWM of the measured concentration. The validity criteria were fulfilled.

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
2015-05-11 to 2015-05-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
QSAR value. The substance falls into applicability domains of the model QSAR.
Justification for type of information:
QSAR prediction
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
Remarks:
(QSAR model)
Principles of method if other than guideline:
The chronic toxicity of the daphnids was determined using a validated QSAR for the Mode of Action in
question, (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 30 chemicals
derived from 21-day test on daphnids, for which the concentrations of the test item had been determined by chemical
analyses over the test period.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
not applicable
Test organisms (species):
Daphnia magna
Test type:
other: QSAR model
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
21d-NOEC
Post exposure observation period:
not applicable
Hardness:
The QSAR is based on data from studies performed at acceptable hardness to ensure control survival.
Test temperature:
The QSAR is based on data from studies performed at between 20 - 23 °C.
pH:
The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0.
Dissolved oxygen:
The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%).
Salinity:
The QSAR is based on data from studies performed on freshwater species.
Nominal and measured concentrations:
The QSAR is based on data from studies performed using measured concentrations or with acceptable stability.
Details on test conditions:
not applicable
Reference substance (positive control):
no
Remarks:
(QSAR model)
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.05 mg/L
Conc. based on:
test mat.
Basis for effect:
reproduction
Remarks on result:
other: [0.035-0.070]
Details on results:
not applicable
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
95% CL [0.035-0.070] QSAR statistical parameters are given in the QMRF and QPRF.

no data

Validity criteria fulfilled:
yes
Remarks:
(The substance falls into applicability domains of the model QSAR.)
Conclusions:
21d-NOEC for d-limonene is 0.050 mg test item/L with 95%-Confidence Limit of 0.035-0.070 mg test item/L.
Executive summary:

The chronic toxicity of the daphnids was determined using a validated QSAR for the Mode of Action in question (MOA 1, non-polar narcosis). The QSAR is based on validated data for a training set of 30 chemicals derived from 21-day test on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The chronic toxicity of d-limonene to daphnids has been investigated using a QSAR model that predicts reduction of daphnid reproduction in an OECD 211 study. d-Limonene, falls within the applicability domain of the model as demonstrated in the QPRF.

The 21d-NOEC was predicted as 0.050 mg/L with 95%-Confidence Limit between 0.035 and 0.070 mg /L. The NOEC was based on reproduction. 

This toxicity study is acceptable and can be used for that endpoint.

 

Description of key information

The substance exhibits a 21-day EC10 of 0.153 mg/L for freshwater invertebrates.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater invertebrates:
0.153 mg/L

Additional information

One experimental data and a valid QSAR prediction are available and are provided as endpoint study records.

The experimental study investigated the effects of substance d-limonene on the reproduction of freshwater Daphnia magna during 21 days. The study was performed according to OECD test guideline 211 in a semi-static exposure system. The endpoint assessed is the number of living offspring per surviving female. A concentration-effect relationship was clearly observed. The endpoint values (NOEC, LOEC, EC10, EC50) were based on measured concentrations in abiotic groups that were run in parallel to the test as it was not possible to acquire meaningful results by sampling directly from the beakers containing daphnids. Thus, the results acquired of measured concentrations may be slightly higher than the “true” concentrations to which the daphnids were exposed during 21 days. The EC10 and EC50 were 153 µg/L (95% CL 83 -222 µg/L) and 188 µg/L (95% CL 118 -257 µg/L), respectively. The NOEC was 80 µg/L and the LOEC was 173 µg/L.

Besides, an endpoint value from High Accuracy QSARs is also available. This provides a 21-day NOEC of 50 µg/L (reproduction).

The EC10 value from experiment is obtained by an appropriate statistical method for modelling the concentration/response relationship. Besides, the NOEC is a test concentration for which the substance is observed to have no statistical difference effect by comparison with the control, and thus is being subjective to choice of nominal test concentrations. The NOECs and the LOECs straddle the classification threshold, with NOEC below 0.1 mg/L, but the geometric mean (MATC) of the NOECs and LOECs of the three studies is greater than 0.1 mg/L suggesting that the “true” effect concentration is higher than this classification threshold.

For the purposes of setting a self-classification and defining a key value for chemical safety assessment, the EC10 for long-term toxicity to aquatic invertebrates is used preferentially to NOEC for the same endpoint and the same duration.