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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th January to 19th September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Version / remarks:
Minor deviations from the guideline were observed but these did not affect the quality of the study and the results obtained
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Composition:
Cardanol, saturated side chain (C21H36O) typical content <1%
Cardanol monoene (C21H34O) typical content 26%
Cardanol diene (C21H32O) typical content 16%
Cardanol triene (C21H30O) typical content 36%
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 1.0, 1.8, 3.2, 5.6 and 10 mg/l Water Soluble Fractions
- Sampling method:
- Sample storage conditions before analysis: The samples were analyzed on the day of sampling
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water Soluble Fractions (WSF) of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were prepared individually using M7 media. A three-day period of magnetic stirring was applied to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were allowed to settle for a period of one hour. Thereafter, the WSFs were collected siphoning through glass wool and used as the test concentrations.
- Eluate: Not relevant
- Controls: M7 media
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not relevant
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No evidence of undissolved material
- Other relevant information: None
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Crustacea, Cladocera) (Straus, 1820)
- Justification for species other than prescribed by test guideline: Not relevant
- Age at study initiation (mean and range, SD): <24 hours old
- Weight at study initiation (mean and range, SD): Not measured
- Length at study initiation (length definition, mean, range and SD): Not measured
- Stage and instar at study initiation: Neonates (<24 hours old)
- Valve height at study initiation, for shell deposition study (mean and range, SD): Not relevant
- Peripheral shell growth removed prior to test initiation: Not relevant
- Method of breeding: Cyclical parthenogenesis using specific breeding conditions
- Source: In-house laboratory culture with a known history
- Age of parental stock (mean and range, SD): At least third generation
- Feeding during test: Yes
- Food type: A Chlorella sp. suspension where the daily ration corresponded to 0.2 mg C/ Daphnia/day, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline 211
- Amount: An amount of 0.25 mL of a Chlorella sp. suspension was fed daily except on weekend days when an amount of 0.50 mL was added in one single feed
- Frequency: Twice daily except on weekend days when a single feed each day was provided

ACCLIMATION
- Acclimation period: Not relevant
- Acclimation conditions (same as test or not): Not relevant
- Type and amount of food: Not relevant
- Feeding frequency: Not relevant
- Health during acclimation (any mortality observed): Not relevant

QUARANTINE (wild caught)
- Duration: Not relevant
- Health/mortality: Not relevant

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
To initiate the test, young daphnids < 24 hours old were selected, from parental daphnids greater than two weeks old. Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood. In the parental cultures half of the medium was replaced twice a week after 7 days of cultivation. The cultures were maintained at 18-22°C and were fed daily, a suspension of fresh water algae.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Remarks on exposure duration:
Test solutions were renewed three times a week during the study
Post exposure observation period:
None
Hardness:
Total hardness 179 - 196 mg CaCO3 per litre
Test temperature:
Temperatures in the range of 19-21°C and constant within 2°C
pH:
pH in the range 7.7-8.5 and constant within 1.5 units
Dissolved oxygen:
Oxygen concentration 7.3 – 9.7 mg/L
Salinity:
Not relevant
Conductivity:
Not relevant
Nominal and measured concentrations:
Nominal concentrations of test substance of 1.0, 1.8, 3.2, 5.6 and 10 mg/L based on loading rates in the individually prepared Water Soluble Fractions.
For the measured concentrations variation was observed between the defined time points of the freshly prepared samples, i.e. days 0, 7, 12 and 17. Measured concentrations of cardanol-triene (m/z 297) ranged between 0.43 and 53 µg/L and could not be related to the initial loading rates of the UVCB, i.e. the measured concentrations were not loading rate- related. However, the initial concentrations were generally higher than in the concentrations measured during the range-finding test. Measured concentrations of cardanol-diene (m/z 299) and cardanol-monoene (m/z 301) were similar to the concentrations of cardanol-triene (m/z 297). All concentrations decreased during the renewal periods. Given these results the TWM concentrations were calculated. The TWM concentrations for all three cardanol forms showed a loading rate- related curve, with measured concentrations increasing with the nominal WSF level. TWM concentrations of cardanol-triene, cardanol-diene and cardanol-monoene in the highest WSF were 4.2, 5.1 and 6.3 µg/L respectively.
Details on test conditions:
TEST SYSTEM
- Test vessel: All glass vessel of 60 ml volume (6 x Ø 3.5 cm)
- Type (delete if not applicable): Closed covered with a Perspex plate
- Material, size, headspace, fill volume: Minimum of 50 mL test medium
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter): Not relevant
- Renewal rate of test solution (frequency/flow rate): Semi static, with renewal of test solutions three times a week
- No. of organisms per vessel: One
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20
- No. of vessels per vehicle control (replicates): Not relevant
- Biomass loading rate: No data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7
- Total organic carbon: <0.1 mg Total Dissolved Organic Carbon/L in test medium at the start of the test
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/Mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: No difference as both culture and test medium were M7
- Intervals of water quality measurement:
Temperature, oxygen and pH: At the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids
Hardness: Once a week in fresh and old media from the control and the highest test concentration (10 mg/L)
Light: At the start and the end of the test

TEST SYSTEM
- Test vessel: All glass vessel of 60 ml volume (6 x Ø 3.5 cm)
- Type (delete if not applicable): Closed covered with a Perspex plate
- Material, size, headspace, fill volume: Minimum of 50 mL test medium
- Aeration:
- Type of flow-through (e.g. peristaltic or proportional diluter): Not relevant
- Renewal rate of test solution (frequency/flow rate): Semi static, with renewal of test solutions three times a week
- No. of organisms per vessel: One
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20
- No. of vessels per vehicle control (replicates): Not relevant
- Biomass loading rate: No data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7
- Total organic carbon: <0.1 mg Total Dissolved Organic Carbon/L in test medium at the start of the test
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/Mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: No difference as both culture and test medium were M7
- Intervals of water quality measurement:
Temperature, oxygen and pH: At the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids
Hardness: Once a week in fresh and old media from the control and the highest test concentration (10 mg/L)
Light: At the start and the end of the test

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment
- Photoperiod: 16 hours light: 8 hours dark
- Light intensity: Light intensity did not exceed 1,000-1,500 lux (measured values 599 to 770 lux during the test)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Parental daphnids
- Mortality (immobility) of parental daphnids: at the end of the test
- Presence of eggs in the brood pouch: every work day
- Individual body length: at the end of the test
Offspring
- Appearance first brood: when observed
- Newborn daphnids: Every workday, the number of newborn young was counted and the condition of the young recorded. Thereafter the young were removed
- Presence of unhatched eggs: when observed
- Incidence of immobility: when observed

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8 times
- Justification for using less concentrations than requested by guideline: Not relevant
- Range finding study: Yes
- Test concentrations: Water Soluble Fractions based on loading rates of 0.10, 1.0 and 10 mg/L of the test substance were tested. A control group was also included. Each concentration consisted of two replicates containing a total of ten daphnids (five in each replicate). The total test period was ten days by which time the first brood should had been produced in all exposure groups.
- Results used to determine the conditions for the definitive study: On the last day of range finding study (Day 10) two parental daphnids were found dead in one replicate of the WSF at 10 mg/L. Both the surviving and the dead daphnids in this replicate were observed under a microscope and were all found to be covered with test substance. Hence, the observed mortality in this replicate could be related to mechanical hampering rather than intrinsic toxicity. The other replicate of the WSF at 10 mg/L showed no parental mortality, but did have some aborted eggs on the last day of exposure. No parental mortality, immobile young or aborted eggs were observed in the control and in the WSFs at 0.10 and 1.0 mg/L. On this basis a concentration series of 1.0 to 10 mg/L was used in the definitive study.
Reference substance (positive control):
no
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rate
Basis for effect:
mortality: Number of living offspring produced per surviving parental animal {for Daphnia magna, TG 211}
Remarks on result:
other: No significant effects on any of the measured parameters for parental daphnids and offspring at any of the WSFs including the highest concentration of 10 mg/l
Details on results:
- Behavioural abnormalities: None observed
- Observations on body length and weight: No statistically significant reduction of growth (based on the length of individuals at the end of the study) was found at any of the test concentrations
- Other biological observations:
In the controls, the presence of eggs in the brood pouch was recorded for the first time on day 4-5 and the first brood appeared on day 8. On average the first recording of young was similar to the control in all the test concentrations.
On average, 150 young were produced per surviving daphnid in the control. Statistical analysis showed that the reproduction of the daphnids was not significantly different from the control group at any of the test concentrations.
- Mortality of control: One out of the nineteen parental daphnids exposed to the control died (i.e. was immobile) during the test period and parental mortality did not exceed 20% in the control, with the actual value being 5.3%.
- Other adverse effects control:
- Immobilisation of control: One out of the nineteen parental daphnids exposed to the control was immobile during the test period.
- Abnormal responses: No abnormal responses observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No, all WSF’s were observed to be clear directly after preparation. Further, no undissolved material was observed during the exposure
- Effect concentrations exceeding solubility of substance in test medium: The 21d-NOEC was above the solubility limit of the UVCB in water (0.305 mg/l) based on nominal levels, but was corrected based on the measured data for the three forms of cardanol (triene, diene and monoene).
Results with reference substance (positive control):
No positive control used
Reported statistics and error estimates:
The values for reproduction observed at various concentrations of the test substance were expressed as mean number of living young per surviving parent. The cumulative values for reproduction at each concentration were compared to those recorded in the control (day 21 of exposure). Further, the length of the parental daphnids (day 21) exposed to the test substance were compared to those of the control organisms.

Mortality of parental daphnids was analysed with Fisher`s exact binomial test with Bonferroni correction (one-sided greater, alpha = 0.05) and qualitative trend analysis by contrasts (monotonicity of concentration/response).

The following statistical procedures were used for the reproduction and growth data:

• Data distribution: Shapiro-Wilk´s Test
• Homogeneity of variance: Levene´s Test (with Residuals)

Differences between treatments and the control for reproduction: Dunnett’s Multiple t-test Procedure (one-sided smaller, alpha = 0.05) after trend analysis by contrasts (monotonicity of concentration/response).

Differences between treatments and the control for growth: Williams Multiple Sequential t-test Procedure (one-sided smaller, alpha = 0.05) after trend analysis by contrasts (monotonicity of concentration/response).

All analyses were performed with ToxRat Professional 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1        Summary of the analytical results measured in the OECD TG211Daphnia magnaReproduction Test

 

Water Soluble Fraction (WSF) loading rate

Analytical data for the different forms of cardanol measured (µg/L)

Cardanol triene

(m/z 297)

Cardanol diene

(m/z 299)

Cardanol monoene

(m/z 301)

Mean in fresh samples

TMW

Mean in fresh samples

TWM

Mean in fresh samples

TMW

1.0

10.4

1.4

12.5

2.1

15.1

3.2

1.8

13.2

2.1

15.4

2.8

18.0

3.7

3.2

23.2

3.1

25.3

4.0

28.8

5.3

5.6

22.9

3.5

24.9

4.5

27.2

5.7

10.0

29.5

4.2

29.0

5.1

30.0

6.3

Table 2       Summary of the results for the endpoints measured in the OECD TG211 Daphnia magna Reproduction Test

 

Water Soluble Fraction (WSF) loading rate

Parental immobility (%)

Cumulative offspring per surviving parent at Day 21

Body length at Day 21 (mm)

Mean number

% change

Mean length

% change

Control

5.3

149.8

-

4.50

-

1.0

11.1

163.8

+9.3

4.59

+2.1

1.8

30.0

164.0

+9.5

4.42

-1.8

3.2

11.1

162.8

+8.7

4.52

+0.6

5.6

0.0

161.4

+7.8

4.45

-1.0

10.0

0.0

155.8

+4.0

4.42

-1.7

 

 

Validity criteria fulfilled:
yes
Remarks:
The mortality of the parent animals (female Daphnia) in the controls did not exceed 20% at the end of the test (actual value 5.3%). AND The average cumulative number of young per female in the controls after 21 days was ≥ 60 (actual value 150 ± 9%).
Conclusions:
A semi-static reproduction test was performed to OECD Guideline 211 in which neonates (<24 hour old) of Daphnia magna were exposed to individual WSFs prepared at loading rates of 1.0 to 10 mg/L, Distilled Grade (Cashew Nutshell Extract, Decarboxylated, Distilled). The test substance did not affect parental mortality (immobility) and reproduction (offspring per surviving daphnid) and the growth of the offspring (based on individual length at the end of the study). This resulted in a NOEC, based on nominal loading rate, of 10 mg/L.

Time weighted mean concentrations of cardanol-triene, cardanol-diene and cardanol-monoene in the highest WSF (10 mg/L) were 4.2, 5.1 and 6.3 µg/L, whilst the mean concentrations of the three forms in freshly prepared solutions were 29.5, 29.0 and 30.0 µg/L. Based on the measured data for the three forms of cardanol the NOEC translated to a test substance concentration of 113.5 µg/L.
Executive summary:

The OECD TG211 test was conducted to evaluate the effects of Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) on the mobility, the growth and the reproductive output ofDaphnia magna. For this purpose, test organisms were exposed to aqueous solutions containing the test substance at various concentrations. The time of the first production of young, the number of young born, immobility and other signs of intoxication observed were compared with corresponding parameters in the control organisms.

 

The batch of Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) tested was a viscous straw coloured to dark brown liquid. The test substance was a UVCB and not completely soluble in test medium at the loading rates initially prepared. Water Soluble Fractions (WSFs) of the test substance were individually prepared at loading rates of 1.0, 1.8, 3.2, 5.6 and 10 mg/L and used as the test concentrations.

 

The definitive reproduction test was performed based on the results of a preceding 10-day range-finding test. It was performed in a semi-static system, which included 10 vessels per test concentration and 20 vessels for the control group. Each of the vessels contained one neonate (<24h old)Daphnia magnain 50 mL of the M7 test medium. The study duration was 21 days and the test solutions were renewed three times a week. The daphnids were fed on a daily basis with aChlorella pyrenoidosasuspension. Every workday the condition of the parental daphnids was recorded, while during the reproduction phase the number of living offspring, immobile young and appearance of unhatched (aborted) eggs was recorded. At the end of the test the length of the surviving parental daphnids was measured. The study met the acceptability criteria prescribed in the Test Guideline and was considered valid.

 

During the test samples for analyses were taken at the start and the end of two intervals of 72 hours (nominal days 0 and 3, 7 and 10) and two intervals of 48 hours (nominal days 12 and 14, 17 and 19). In each sample, analysis was performed on three forms of cardanol, i.e. cardanol-triene (m/z 297), cardanol-diene (m/z 299) and cardanol-monoene (m/z 301), which are the major constituents of the UVCB.

 

Variation in the concentrations of the different forms of cardanol was observed between the defined time points of the freshly prepared samples, i.e. days 0, 7, 12 and 17. Measured concentrations of cardanol-triene (m/z 297) ranged between 0.43 and 53 µg/L and could not be related to the initial loading rates of the UVCB, i.e. the measured concentrations were not loading rate-related. However, the initial concentrations were generally higher than in the concentrations of this form measured during the range-finding test. Measured concentrations of cardanol-diene (m/z 299) and cardanol-monoene (m/z 301) were similar to the concentrations of cardanol-triene (m/z 297). All concentrations decreased during the renewal periods. Given these results the TWM concentrations were calculated. The TWM concentrations for all three cardanol forms showed a loading rate-related curve, with measured concentrations increasing with the nominal WSF level. Time weighted mean concentrations of cardanol-triene, cardanol-diene and cardanol-monoene in the highest WSF (10 mg/L) were 4.2, 5.1 and 6.3 µg/L, whilst the mean concentrations of the three forms in freshly prepared solutions were 29.5, 29.0 and 30.0 µg/L.

 

In the study Distilled Grade (Cashew Nutshell Extract, Decarboxylated, Distilled) did not affect parental mortality (immobility) and reproduction (offspring per surviving daphnid) and the growth of the offspring (based on individual length at the end of the study). This resulted in a NOEC based on nominal loading rate of 10 mg/L. However, this translated to a test substance concentration of 113.5 µg/L based measured data for the three forms of cardanol.

Description of key information

A semi-static reproduction test was performed to OECD Guideline 211 in which neonates (<24 hour old) of Daphnia magna were exposed to individual WSFs prepared at loading rates of 1.0 to 10 mg/L, Distilled Grade (Cashew Nutshell Extract, Decarboxylated, Distilled). The test substance did not affect parental mortality (immobility) and reproduction (offspring per surviving daphnid) and the growth of the offspring (based on individual length at the end of the study). This resulted in a NOEC, based on nominal loading rate, of 10 mg/L.

 

Time weighted mean concentrations of cardanol-triene, cardanol-diene and cardanol-monoene in the highest WSF (10 mg/L) were 4.2, 5.1 and 6.3 µg/L, whilst the mean concentrations of the three forms in freshly prepared solutions were 29.5, 29.0 and 30.0 µg/L. Based on the measured data for the three forms of cardanol the NOEC translated to a test substance concentration of 113.5 µg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
113.5 µg/L

Additional information