No IUPAC name is currently defined for Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled (“Distilled Residue Grade”).

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2nd July to 1st October 2020

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)

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch (Lot) Number: FP-0306
Expiry date: 31st December 2021
Physical Description: Straw coloured to dark brown liquid
Purity/Composition: 100% (UVCB)
Composition: Cardanol, saturated side chain (C21H36O) typical content <2%
Cardanol monoene (C21H34O) typical content 11%
Cardanol diene (C21H32O) typical content 7%
Cardanol triene (C21H30O) typical content 15%

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: Aliquots of freshly prepared and old samples were collected
- 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 Elendt M7 media. A one-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 by siphoning through a glass wool plug and one sheet of filter paper before being used as the test concentrations.
- Eluate: Not relevant
- Differential loading: Not relevant
- Controls: Elendt 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)
- Strain/clone: No information
- 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: Each daphnid received an algal suspension (Raphidocelis subcapitata) daily throughout the test along with GEMMA Micro 300 fish food suspension provided on Days 0, 1, 2 and 3 of the test.
- Amount: Each daphnid received between 5 and 20 µL of an algal suspension daily throughout the test with 20 µL of GM300 fish food suspension provided on Days 0, 1, 2 and 3 of the test. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals. Equal amounts of food were given to each daphnid.
- Frequency: Once daily

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:
Adult Daphnia were maintained in 150 mL glass vessels containing Elendt M4 medium in a temperature controlled room at approximately 20 oC. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (Raphidocelis subcapitata and GEMMA Micro 300 fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Remarks on exposure duration:

Test solutions were renewed daily during the study

Post exposure observation period:

None

Hardness:

Total hardness 246 to 276 mg CaCO3 per litre in the control and highest test group throughout the test

Test temperature:

Temperatures in the range of 19-22°C throughout the test

pH:

pH in the range 7.1-8.5 and constant within 1.5 units

Dissolved oxygen:

Oxygen concentration 8.3 – 9.3 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.
The analysis of the exposure solutions from the test provided information on the concentrations of the three forms of cardanol, namely the triene (m/z 297), diene (m/z 299) and monoene (m/z 301) (which are the major non-polymeric constituents of the UVCB), at various points throughout the 21-day study. There was analysis of the fresh samples prepared on Days 0. 6, 13 and 20. Old samples taken on Days 0, 7, 14 and 21 (after 24 hours exposure) were also analysed. Variations in the concentrations of the different forms of cardanol were observed between the defined time points of the freshly prepared samples, i.e. days 0, 6, 13 and 20. In the different WSFs the mean measured concentrations of cardanol triene were higher than those of cardanol diene, with those of cardamol monoene being the lowest. The mean measured concentrations of each of the different forms of cardanol were generally similar in the 1.0 to 5.6 mg/l loading rate WSFs, but were higher in the 10 mg/l WSF. All concentrations decreased during the renewal periods. Given these results the Time Weighted Mean (TWM) exposure concentrations were calculated, which for all three cardanol forms showed a loading rate-related curve, with measured concentrations generally increasing with the nominal WSF level.

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass vessel of 150 ml volume
- Type (delete if not applicable): Closed covered with a plastic lid
- Material, size, headspace, fill volume: 100 mL test medium
- Aeration: No aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): Not relevant
- Renewal rate of test solution (frequency/flow rate): Semi static, with daily renewal of test solutions
- No. of organisms per vessel: One
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): Not relevant
- Biomass loading rate: No data

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt M7
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: 0.9 mmol/L
- Ca/Mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: No difference as both culture and test medium were Elendt M7
- Intervals of water quality measurement:
Temperature, oxygen and pH: Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The pH and dissolved oxygen concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature was measured using a digital thermometer. Measurements were made on one replicate for each test concentration.
Hardness: Once a week in fresh and old media from the control and the highest test concentration (10 mg/L)
Light: Daily throughout the test

OTHER TEST CONDITIONS
- Adjustment of pH: No adjustment
- Photoperiod: 16 hours light: 8 hours dark with 20 minute dawn and dusk transitional periods
- Light intensity: Light intensity not to exceed 1,500 lux (measured values 879 to 1007 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 day, 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 five replicates containing a single daphnid. 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: No immobilization or sub-lethal effects of exposure were observed at the test concentrations of 1.0 and 10 mg/L loading rate WAF. However, 20% immobilization was observed at 0.1 mg/L loading rate WAF. Sub lethal effects of exposure were observed at test concentrations of 10 mg/L loading rate WAF. The response (in the adults) was pale in colouration when compared to the controls. Based on this information loading rates WAFs of 1.0, 1.8, 3.2, 5.6 and 10 mg/L were selected for the definitive test.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate

Basis for effect:

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, 168.5 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: For the parental mortality (immobility) endpoint two inadvertent mortalities occurred in the controls along with the one each in the 1.0, 5.6 and 10 mg/l WSFs. However, statistical analysis showed that mortality (immobility) was not different from the control group at any of the test concentrations
- Other adverse effects control:
- Immobilisation of control: For the parental mortality (immobility) endpoint two inadvertent mortalities occurred in the controls
- Abnormal responses: No abnormal responses observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- 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):

- Results with reference substance valid? No positive control used
- Relevant effect levels: No data
- Other: Not relevant

Reported statistics and error estimates:

For the determination of the "Lowest Observed Effect Loading Rate" (LOEL) and the "No Observed Effect Loading Rate" (NOEL) for the number of parental generation Daphnia mortalities for the control and each test group was estimated by inspection of the mortality data. For reproduction, the numbers of live young produced per surviving adult over the duration of the test for the control and each test group were compared using the Multiple Sequentially-Rejective Median (2 x 2-Table) test after Bonferroni-Holm incorporating Shapiro Wilk's test on Normal Distribution and Cochran’s test Procedure on Variance Homogeneity. Results from the control and each test group Daphnia length data, determined for the surviving daphnids on termination of the test, were compared using Dunnett’s Multiple t-test Procedure incorporating Shapiro Wilk's test on Normal Distribution and Levene’s test on Variance Homogeneity.

All statistical analyses were performed using the ToxRatPro software package (TOXRAT).

Table 1          Summary of the analytical results measured in the OECD TG211 Daphnia magna Reproduction 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	10.8	3.25	5.1	1.91	2.9	1.01
1.8	7.0	2.35	3.3	1.36	1.6	0.61
3.2	10.7	3.26	5.1	1.98	2.5	0.91
5.6	10.2	3.61	4.9	2.18	2.3	1.04
10.0	12.3	4.35	6.5	2.91	3.6	1.47

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^1(%)	Cumulative offspring per surviving parent at Day 21		Body length at Day 21 (mm)
		Mean number	% change	Mean length	% change
Control	0 (20)	168.5	-	4.26	-
1.0	0 (10)	185.1	+9.9	4.10	-3.8
1.8	0	175.7	+4.3	4.16	-2.4
3.2	0	177.9	+5.6	4.07	-4.5
5.6	0 (10)	171.2	+1.6	4.08	-4.3
10.0	0 (10)	163.4	-3.0	4.03	-5.4

Note:¹–Data oninadvertent parental mortality (immobility) are shown in parentheses

 

Validity criteria fulfilled:

yes

Conclusions:

The tests validity criteria were fulfilled as:
• The mortality of the parent animals (female Daphnia) in the controls did not exceed 20% at the end of the test (actual value 0% but with 20% inadvertent mortalities).
• The average cumulative number of young per female in the controls after 21 days was ≥ 60 (actual value 168.5 ± 12.2%).

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 of Distillation Residue Grade (Cashew Nutshell Extract, Decarboxylated, Distillation Residue). The test substance did not affect parental mortality (immobility), reproduction (offspring per surviving daphnid) or the growth of the parents (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 4.35, 2.91 and 1.47 µg/L, whilst the mean concentrations of the three forms in freshly prepared solutions were 12.3, 6.50 and 3.60 µg/L. As a result of the absence of effects the actual NOEC based on loading rate could be considerably higher. Therefore, in order to avoid generating an overly precautionary NOEC it was based on the mean measured concentrations of the different forms of cardanol from freshly prepared samples of the 10 mg/l WSF rather than the time weighted mean values in this WSF. Based on the measured data for the three forms of cardanol the NOEC (loading rate) translated to a test substance concentration of 67.9 µg/L.

Executive summary:

The OECD TG211 test was conducted to evaluate the effects of Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) on the mobility (mortality), the growth and the reproductive output of Daphnia 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, Distillation Residue (Distillation Residue 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. There was daily replacement of the test solutions.

 

The definitive reproduction study was performed based on the results of a preceding 10-day range-finding test. In the study,groups of 10 neonates (<24 hr old) were exposed the series of individually prepared Water Soluble Fractions (WSFs). Each daphnid received between 5 and 20 μl of an algal suspension (Raphidocelis subcapitata) daily throughout the test with 20 μl of GEMMA Micro300 fish food suspension provided on Days 0, 1, 2 and 3 of the test. Feeding was at a level of approximately 0.1 to 0.2 mg carbon/daphnid/day, dependent on the age and size of the animals, which is the recommended value for daily feeding per daphnid in the reproduction test according to the OECD Guideline. Equal amounts of food were given to each daphnid.

The analysis of the exposure solutions from the test provided information on the concentrations of the three forms of cardanol, namely the triene (m/z 297), diene (m/z 299) and monoene (m/z 301) (which are the major non-polymeric constituents of the UVCB), at various points throughout the 21-day study. There was analysis of the fresh samples prepared on Days 0. 6, 13 and 20. Old samples taken on Days 0, 7, 14 and 21 (after 24 hours exposure) were also analysed. Variations in the concentrations of the different forms of cardanol were observed between the defined time points of the freshly prepared samples, i.e. days 0, 6, 13 and 20. In the different WSFs the mean measured concentrations of cardanol triene were higher than those of cardanol diene, with those of cardamol monoene being the lowest. The mean measured concentrations of each of the different forms of cardanol were generally similar in the 1.0 to 5.6 mg/l loading rate WSFs, but were higher in the 10 mg/l WSF. All concentrations decreased during the renewal periods. Given these results the Time Weighted Mean (TWM) exposure concentrations were calculated, which for all three cardanol forms showed a loading rate-related curve, with measured concentrations generally increasing with the nominal WSF level.

 

In the study exposure to Distillation Residue Grade did not affect reproduction, parental mortality and growth of juvenileDaphnia magnaexposed to WSFs prepared at the loading rates of 1.0 to 10 mg/l (see Table 2), resulting in a NOELR of 10 mg/l based on loading rate.

 

For the parental mortality (immobility) endpoint two inadvertent mortalities occurred in the controls along with the one each in the 1.0, 5.6 and 10 mg/l WSFs. However, statistical analysis showed that mortality (immobility) was not different from the control group at any of the test concentrations (see Table 2).

 

In the control, the presence of eggs in the brood pouch was recorded for the first time on day 4-5 and the first brood appeared in the controls on day 8. The first recording of young was similar to the controls in all the test concentrations. There were 15 aborted eggs observed in the control group and 11 aborted eggs observed in the 1.8 mg/l loading rate test group on Day 11.

 

At the end of the test, 169 young on average were produced per surviving daphnid in the controls. Statistical analysis showed that the reproduction of the daphnids was not significantly different from the control group at any of the test concentrations (see Table 2).

 

The mean body length of the surviving daphnids per concentration measured at the end of the test was statistically significantly reduced in all the WSFs compared to the controls except that of the 1.8 mg/l WSF loading rate (see Table 2). However, after a review of the historical control data which showed that all body weights were within the historical control data range and as the decrease in body length compared to the controls was limited to a maximum of 5% and a true dose relationship was not apparent for this parameter it is considered that these differences are not biologically significant.

 

Based on the data for the different endpoints a No Observable Effect Loading Rate (NOELR) of 10 mg/l has been determined. Time weighted mean concentrations of cardanol-triene, cardanol-diene and cardanol-monoene in the highest WSF (10 mg/L) were 4.35, 2.91 and 1.47 µg/L, whilst the mean concentrations of the three forms in freshly prepared solutions were 12.3, 6.50 and 3.60 µg/L. As a result of the absence of effects the actual NOEC based on loading rate could be considerably higher. Therefore, in order to avoid generating an overly precautionary NOEC it was based on the mean measured concentrations of the different forms of cardanol from freshly prepared samples of the 10 mg/l WSF rather than the time weighted mean values in this WSF. Based on the measured data for the three forms of cardanol the NOEC translated to a test substance concentration of 67.9 µg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

67.9 µg/L