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Long-term toxicity to fish

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Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15-23 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline No. 212. All validity criteria were fulfilled.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 212 (Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages)
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): 0.843 g/mL
- Water solubility: 4.0-5.7 mg/L
- Vapour pressure (298 °K): 200 Pa
Analytical monitoring:
yes
Details on sampling:
For all test concentrations and the control, samples for analysis were collected from additional test flasks without eggs 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 were collected of each test concentration at the initiation of the test, before and after each renewal and at the termination of the test. The samples were collected in 40-mL glass vials and stored at -20 ± 2.0 °C. Samples relevant for the calculation of the LCx, NOEC and LOEC concentrations were sent frozen to the analytical laboratory. Although duplicate samples were collected from each test concentration, 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 1 g/L was prepared by adding 2.65 mL (corresponding to 2.23 g) of the test item to 2.22 L test medium. The following procedure was used:
A 2-liter glass bottle with a magnet (total content 2.22 L) was filled with ISO medium withholding 5-10 mL of the total content. A pipette tip was saturated by filling it 3 times with the test item and 2.65 mL was carefully dosed to the middle of the water phase. The bottle was rapidly filled totally with ISO medium and sealed tightly with a screw cap with PTFE packing. The bottle was covered with black cloth and left for gentle stirring, approximately 350 rpm, at room temperature for 24 ± 2 h. The stirring was stopped and approximately 1750 mL of the mid fraction syphoned off using a silicone 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. The mid fraction (stock solution B) was carefully mixed well without shaking. pH was measured to be 7.9 and no adjustment was therefore necessary.
The test solutions were prepared individually by immediately diluting stock solution B in test medium.
At each renewal, the entire procedure was repeated when preparing fresh test solutions every third day. The pH in stock solution B was 7.8, 7.9 and 8.0, respectively, on days 0, 3 and 6. As pH in the stock solutions were within 7.8 ± 0.5, no pH adjustment was necessary.
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Source: Newly fertilized fathead minnow eggs (Pimephales promelas) obtained from a group of parental fish were delivered by the Institute for the Environment, Brunel University, Kingston Lane, Uxbridge UB8 3PH, England.
- Upon arrival and just before initiation of the test, the embryo developmental stage was determined and it was verified by photography that the embryos were still in the blastodisc cleavage stage.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
8 d
Remarks on exposure duration:
4 days post hatch
Post exposure observation period:
None
Hardness:
Not reported
Test temperature:
25 ± 2.0 °C (min.-max. measured values: 23.5-25.6 °C)
pH:
min.-max. measured values: 7.4-8.3
Extreme values 6.5-6.9 were measured at one occasion in old solution from the highest test concentration where all organisms died in all replicates
Dissolved oxygen:
min.-max. measured values: 69-100 per cent of the air saturation value (ASV)
Extreme value 50% ASV was measured at one occasion in old solution from the highest test concentration where all organisms died in all replicates
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 2.5, 5.3, 11.0, 23.2 and 48.6 % of a saturated solution of the test item in test medium
Details on test conditions:
TEST SYSTEM
- Test vessel: Test was performed in 100-mL Pyrex glass flasks sealed with PTFE-coated screw caps.
- At the initiation of the test, 108 mL of test solution and 10 embryos in the blastodisc cleavage stage were added to each test flask. A minor head space was present in the test flasks and the flasks were shaken gently during the test.
- Thirty eggs (3 replicates of 10 eggs each) were exposed to each concentration of the test item and the control.
- On day 3 and day 6, approx. 95 % of each test solution was removed by pouring of and carefully replacing with the same amount of freshly prepared test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic medium according to ISO 7346

OTHER TEST CONDITIONS
- Photoperiod: Light/dark period of 16:8 h

EFFECT PARAMETERS MEASURED
- During the test, hatching, survival, abnormal appearance and behaviour were observed and recorded daily.
- Dissolved oxygen concentrations, temperature and pH were measured at the initiation of the test, at each renewal of test solutions and at the termination of the test. Temperature was also measured continuously by thermo logger.
Reference substance (positive control):
no
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
0.37 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: hatching rate
Duration:
8 d
Dose descriptor:
LOEC
Effect conc.:
0.67 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: hatching rate
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: abnormal appearance and behaviour
Duration:
8 d
Dose descriptor:
LOEC
Effect conc.:
0.37 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: abnormal appearance and behaviour
Duration:
8 d
Dose descriptor:
NOEC
Effect conc.:
0.059 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
8 d
Dose descriptor:
LOEC
Effect conc.:
0.19 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
8 d
Dose descriptor:
EC10
Effect conc.:
> 0.37 - < 0.67 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
8 d
Dose descriptor:
EC50
Effect conc.:
> 0.37 - < 0.67 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
8 d
Dose descriptor:
LC10
Effect conc.:
0.32 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: survival
Remarks on result:
other: 95% Cl: 0.21-0.41 mg/L
Duration:
8 d
Dose descriptor:
LC50
Effect conc.:
0.41 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: survival
Remarks on result:
other: 95% Cl: 0.31-0.53 mg/L
Details on results:
- Increased hatching rate at the highest test concentration (0.67 mg/L) (based on calculation of time to 50 % hatch. The data did not allow a statistical evaluation).
- Significant effect on the survival rate (100 % mortality at 0.67 mg/L and a calculated LC50: 0.41 mg/L).
- Slight to moderate effect on the appearance and behaviour at 0.37 mg/L (subjective evaluation; the data did not allow a statistical evaluation).
- Significant effect (Students t-test) on the growth rate, measured as length of the surviving larvae at the end of the test was observed at 0.19 and 0.37 mg/L. NOEC is thus determined to be 0.059 mg/L and LOEC 0.19 mg/L. Data did not allow the calculation of ECx.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
LC10 and LC50 values for the endpoint survival was calculated on the basis of the analytical results, by use of the standard procedure for Probit analysis and NOEC and LOEC values were estimated by use of Students t-test.

Table 6.1.2/1: Results from chemical analyses of d-Limonene

Sampling time

 

T = 0 days (mg/L)

 

T = 3 days - old (mg/L)

 

T = 3 days - new (mg/L)

 

T = 6 days - old (mg/L)

 

T = 6 days - new (mg/L)

 

T= 8 days - old (mg/L)

 

Time weighted mean value (mg/L)

 

Control

 

<0.010

 

<0.010

 

<0.010

 

<0.010

 

<0.010

 

<0.010

 

<0.010

 

5.3 %

0.030

0.060

0.070

0.060

0.060

0.090

0.059

11.0 %

0.13

0.16

0.18

0.29

0.21

0.19

0.19

23.2 %

0.25

0.43

0.34

0.48

0.42

0.35

0.37

48.6 %

0.61

0.70

0.67

0.71*

-

-

0.67

* Sampled at T = 5 days, because all eggs/larvae were observed dead

The 2.5 % solutions were not analysed as they were not relevant for the determination of the EC or NOEC values

Table 6.1.2/2: Mean measured concentration, survival and mean length

Dilution

Time-weighted mean concentration (mg/L)

Survival (N = 30)

Mean length (mm)

Control

< 0.010

22

5.28 

2.5 %

Not analysed

22

5.24

5.3 %

0.059

26

5.28

11.0%

0.19

27

5.08

23.2 %

0.37

19

5.07

 48.6%  0.67  0  0

Validity: 

- Overall hatching success and post-hatch success in the control was greater than or equal to 60 and 70 %, respectively. 

- The dissolved oxygen concentration was between 60 and 100 % of the air saturation value (ASV) throughout the test. 

- At one occasion, however, (one out of 3 replicates in the highest test concentrations at day 5) the dissolved oxygen concentration was 50 %. Due to the need for sealed test containers during the test it was not possible to remove dead eggs and larvae. It is expected that the low oxygen percentage observed was due to decomposition of dead eggs and larvae. As all organisms died in the highest test concentration in all 3 replicates the low oxygen concentration observed in the single replicate is not expected to have any influence of the overall results obtained. 

- The water temperature did not differ by more than ± 1.5 °C between test chambers or between successive days at any time during the test (within the range 25 ± 2.0 °C).

Validity criteria fulfilled:
yes
Conclusions:
The NOEC for hatching, abnormal appearance & behaviour and growth were 0.37, 0.19 and 0.059 mg/L; LOEC for hatching, abnormal appearance & behaviour and growth were 0.67, 0.37 and 0.19 mg/L; LC50 was determined to be 0.41 mg/L (95% Cl: 0.31-0.53 mg/L).
Executive summary:

A short-term toxicity test on embryo and sac-fry stages with Pimephales promelas was performed according to OECD Guideline 212 and in compliance with GLP.

d-Limonene was tested at the following nominal concentrations: 0 (control), 2.5, 5.3, 11.0, 23.2 and 48.6% of a saturated solution of the test item in test medium. The test was carried out as a semi-static test in 100 mL Pyrex glass flasks sealed with PTFE-coated screw caps. Approx. 95% of the test solutions were renewed at day 3 and day 6. At termination of the test the growth of the hatched larvae was determined and during the test, hatching, survival, abnormal appearance and behaviour was observed and recorded daily. The duration of the test was 8 days. The concentrations of the test item were determined by chemical analyses during the exposure period.

                                                                                                         

At the highest test concentration (0.67 mg/L), the hatching rate is increased. Significant effect on the survival rate (100% mortality at 0.67 mg/L). Slight to moderate effects were observed on the appearance and behaviour at 0.37 mg/L. The effect on growth rate, measured as length of the surviving larvae at the end of the test was significant at 0.19 and 0.37 mg/L (both 4% reduction).

 

Therefore, the NOEC for hatching, abnormal appearance & behaviour and growth were 0.37, 0.19 and 0.059 mg/L; LOEC for hatching, abnormal appearance & behaviour and growth were 0.67, 0.37 and 0.19 mg/L; LC50 was determined to be 0.41 mg/L (95% Cl: 0.31-0.53 mg/L) and the LC10 was determined to be 0.32 mg/L (95%CI: 0.21 -0.41 mg/L. The effect on the growth rate observed at 0.19 and 0.37 mg/L was less than 10% and data did not allow the calculation of EC10 and EC50. Bot the EC10 and EC50 for growth are between 0.37 and 0.67 mg/L.

Endpoint:
fish early-life stage toxicity
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 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
yes
Remarks:
(QSAR model)
Principles of method if other than guideline:
The chronic toxicity to fish 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 26 chemicals derived from
14 to 32-day test on fish, 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):
other: fish spp.
Details on test organisms:
not applicable
Test type:
other: QSAR model
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Remarks on exposure duration:
28d-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 21 - 27 °C (depending on the species considered)
pH:
The QSAR is based on data from studies performed at acceptable pH between 6.0 - 8.5
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:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.08 mg/L
Conc. based on:
test mat.
Basis for effect:
other: sublethal effects (e.g. growth and hatchability)
Remarks on result:
other: [0.056-0.11]
Details on results:
not applicable
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
95% CL [0.056-0.11] 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:
28d-NOEC for d-limonene is 0.080 mg test item/L with 95%-Confidence Limit of 0.056-0.11 mg test item/L.
Executive summary:

The chronic toxicity to fish 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 26 chemicals derived from 14 to 32-day test on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period.

The chronic toxicity to fish of d-limonene has been investigated using a QSAR model that predicts fish lethality in an OECD 210 study. d-Limonene falls within the applicability domain of the model as demonstrated in the QPRF.

The 28d-NOEC for d-limonene was 0.080 mg test item/L with 95%-Confidence Limit of 0.056-0.11 mg test item/L. The NOEC was based on sublethal effects, like growth and hatchability.

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

Description of key information

The substance exhibits an estimated EC10 for freshwater fish higher than 0.37 mg/L.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.37 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 short-term toxicity on embryo and sac-fry stages of substance d-limonene on freshwater fish Pimephales promelas. The study was performed according to OECD Guideline 212 under semi-static conditions for 8 days. Hatching, survival, abnormal appearance and behaviour were observed daily. Growth length was measured at 8 days (4 days post hatch). The concentrations of d-limonene were determined by chemical analyses during the exposure period. The analysis of concentrations were performed by sampling analysis from additional test flasks without fish eggs (abiotic groups). At the highest measured concentration of 0.67 mg/L (time weighted mean), the hatching rate is increased. However, 100% mortality was observed at 0.67 mg/L. The effect on growth rate, measured as length of the surviving larvae at the end of the test was significant at 0.19 and 0.37 mg/L (both 4% reduction) but no clear concentration / response was observed for growth endpoint. The endpoint values ​​are based on the measured concentration of d-limonene in abiotic replicates. Thus, the acquired results of measured concentrations may be slightly higher than the “true” concentrations to which the fish were exposed during 8 days. The 8-day EC10 are estimated between 0.37 and 0.67 mg/L.

Besides, an endpoint value from High Accuracy QSARs is also available. This provides a 28-day NOEC of 0.08 (0.056-0.11) mg/L for sublethal effects on fish. The value of NOEC is the test concentration immediately below the LOEC. Where EC10-values are available, these are preferred over NOEC values for the same endpoint.

Justification on the choice of OECD TG 212

In the ECHA Guidance R.7B, 7.8.4.1, Guideline OECD 212 is listed under the long-term fish tests and mentioned as one of the tests to be used for refinement of the PNEC value. The OECD 212 test includes the same sensitive life stages as OECD 210. It misses one stage: where the alevins start feeding by themselves. The current OECD 212 study with d-limonene concludes a NOEC with a statistically significant effect on growth of only 4%. The 4% growth reduction is observed at two consecutive test concentrations (0.19 and 0.37 mg/L), thus there is no dose-effect relation in that section of the curve. The level with 10% effect is actually above 0.37 mg/L, and although the EC10 could not be derived statistically, 0.37 mg/L forms the appropriate basis for the classification. In view of the difficulties in testing this substance (poorly soluble in water, biodegradable, highly volatile, potentially photodegradable) a longer test duration than 7 days would be technically extremely difficult to perform as was already encountered in the daphnid reprotoxicity study due to the problems of solution renewal, the volumes necessary, biodegradation due to bacterial growth etc. This study would be even more complicated to perform than the daphnid test.. Moreover, the test would then include free living vertebrate test species for which prior approval would be needed.