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

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 December 2019 to 10 January 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Conducted under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 218 (Sediment-Water Chironomid Toxicity Test Using Spiked Sediment)

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: 30th April 2020
Physical Description: Straw coloured to dark brown liquid
Purity/Composition: 100% (UVCB)
Composition: Cardanol, saturated side chain (C21H36O) typical content <1%
Cardanol monoene (C21H34O) typical content 11%
Cardanol diene (C21H32O) typical content 7%
Cardanol triene (C21H30O) typical content 15%

Analytical monitoring:

yes

Details on sampling:

SEDIMENT
- Concentrations: Controls (blank and acetone solvent) and nominal exposure concentrations of 62.5, 125, 250, 500 and 1000 mg /kg dry weight test substance (see Table 1)
- Sampling interval: On days 0 and 28 of the test
- Sample storage before analysis: Not applicable, samples were analysed on the day of sampling

PORE WATER
- Concentrations: Measured pore water concentrations from each of the nominal sediment concentrations were <1.58 mg/L (based on cardanol triene, m/z 297 data) and <1.26 mg/L (based on cardanol monoene, m/z 301 data) on Day 0 (see Table 1)
- Sampling interval: On days 0 and 28 of the test
- Sample storage before analysis: Not applicable, samples were analysed on the day of sampling

OVERLYING WATER
- Concentrations: Measured overlying water concentrations from each of the nominal sediment concentrations were <0.211 mg/L (based on cardanol triene, m/z 297 data) and <0.204 mg/L (based on cardanol monoene, m/z 301 data) on Day 0 (see Table 1)
- Sampling interval: On days 0 and 28 of the test
- Sample storage before analysis: Not applicable, samples were analysed on the day of sampling

Vehicle:

no

Test organisms (species):

Chironomus riparius

Details on test organisms:

TEST ORGANISM
- Common name: Chironomus riparius
- Source: In-house laboratory culture with a known history
- Details on collection: Not relevant
- Breeding conditions: Egg masses produced in the continuous in-house culture were used to start a new batch of organisms for the test
- Handling of egg masses and larvae: Four to five days before start of the test (Day 0), egg masses were taken from the culture and deposited into small vessels containing culture medium
- Age of animals at beginning of exposure: On Day 0, twenty larvae of the first larval stage (2 to 3 days old) were allocated randomly to each test vessel with a pipette.
- Feeding during test: Yes
- Food type: Pelleted fish food (Essence, Coppens, Nettental, Germany)
- Amount: In general, on nominal days:
Days 0 to 10: 0.5 mg pelleted fish food per larvae per day
Days 11 to 27: see the table below

Number of larvae in vessel Number of emerged midges Amount of food
(mg/day/ vessel)
20-15 0-5 20
14-10 6-10 15
9-5 11-15 10
4-1 16-19 5
0 20 0
- Frequency: Daily from Day 0 to 27

ACCLIMATION – Not relevant

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Remarks:

Artificial soil substratum prepared according to OECD TG218

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

None

Hardness:

Test range from 214 to 250 mg CaCO3/L

Test temperature:

Test range from 19.7 – 21.3 oC

pH:

Test range from pH 7.2 to 8.6 in the water layer.

Dissolved oxygen:

Test range from 7.1 to 9.1 mg/L, except during the obligatory non-aeration period during and after the addition of the larvae when the minimal dissolved oxygen concentration was 2.5 mg/L.

Salinity:

Not relevant

Ammonia:

Test range varied from 0 to 0.5 mg/L on Day 0 to values from >10 mg/L on Day 28. The corresponding free ammonia concentrations (4% at pH 8) are considered acceptable for Chironomus riparius.

Conductivity:

Not measured

Nominal and measured concentrations:

Nominal sediment exposure concentrations of 62.5, 125, 250, 500 and 1000 mg /kg dry weight test substance along with blank and solvent (acetone) controls were used in the test. At the start of the test the test substance concentration in the sediment was between 76% and 98% of the nominal test concentration. During the test, the percentage of the nominal concentrations decreased. The test substance concentration in the sediment was between 56% and 73% of the nominal test concentration on Day 28. Since measured test substance concentrations, based on the different forms of cardanol, in sediment were not in agreement with nominal concentrations, geometrical means were calculated from the measured exposure concentrations at the start and end of the test.

The test substance concentrations were < 0.2 mg/L in overlying water and < 1.6 mg/L in pore water for the two major forms of cardanol measured (the monoene and triene). The total measured test substance concentration, analysed on the two forms of cardanol, was between 76% and 98% of the nominal initial concentration at Day 0. At the end of the test, the mass balance decreased. The total measured test substance concentration in all exposure phases was between 56% and 73% of the initial level at Day 28.

Measured concentrations of each form of cardanol in the sediment, pore water and overlying water in samples collected from each exposure level are given in Table 1 for Day 0 and Table 2 for Day 28.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): All-glass, 600 ml beakers, ø ca. 8 cm.
- Sediment volume: A layer of ca. 1.5 cm of spiked sediment was added to each test vessel.
- Weight of wet sediment with and without pore water: The total amount of wet sediment was approximately 85 g per test vessel, consisting of 12 g treated sand and 73 g sediment. This corresponds with approximately 60 g dry sediment per vessel.
- Overlying water volume: 270 ml per vessel
- Depth of sediment and overlying water: 6 cm of test water was added to the sediment without considerable disturbance. The ratio of overlying water : sediment was 4 : 1. Test vessels were covered with a nylon mesh and left under test conditions for an equilibrium period of approximately 48 hours before the organisms were added.
- Aeration: Yes
- Aeration frequency and intensity: The test vessels were continuously and gently aerated using a pipette, 2 to 3 cm above the sediment layer, except for a period just after the addition of the larvae.
- Replacement of evaporated test water, if any: If applicable, evaporated water was replaced by Milli-RO water three times a week.

EXPOSURE REGIME
- No. of organisms per container (treatment): Twenty larvae of the first larval stage (2 to 3 days old) were allocated randomly to each test vessel with a pipette.
- No. of replicates per treatment group: Four per group
- No. of replicates per control / vehicle control: Four per group
- Feeding regime: Daily addition of food from Day 0 to 27
- Type and preparation of food: Pelleted fish food
- Amount of food: 0.5 mg of pelleted fish food per larvae per day from Day 0 to 10. On days 11 to 27 the amount of food supplied was adjusted for the number of emerged larvae present.

RENEWAL OF OVERLYING WATER
- Details on volume additions: If applicable, evaporated water was replaced by Milli-RO water three times a week.
- Flow-rate: Not relevant

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): Adjusted ISO medium, formulated using RO water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with a hardness of 180 mg CaCO3 per litre and a pH of 7.7 ± 0.3.
- Source of water (if non-standard medium): Not relevant
- Location: Not relevant
- Description of sampling site: Not relevant
- Filtration: Not relevant
- Alkalinity: Not relevant
- Salinity: Not relevant
- Conductivity: Not relevant
- Particulate matter: Not relevant
- Total organic carbon: Not relevant
- Chemical oxygen demand: Not relevant
- Unionized ammonia: Not relevant
- Residual chlorine: Not relevant
- Total organic chlorine compounds and PCBs: Not relevant
- Total organophosphorous compounds: Not relevant
- Total organic chlorine: Not relevant

SOURCE OF NATURAL SEDIMENT
- Location and description of sampling site: Not relevant
- Contamination history of site: Not relevant

HANDLING OF NATURAL SEDIMENT
- Time of collection: Not relevant
- Core depth: Not relevant
- Water depth: Not relevant
- Storage conditions: Not relevant
- Storage duration (prior to test): Not relevant

CHARACTERIZATION OF ARTIFICIAL SEDIMENT
- % dry weight of sphagnum moss peat: 5.0
- Particle size distribution
- % sand: Not measured
- % silt: Not measured
- % clay: Not measured
- Composition (if artificial substrate): Industrial (silver) sand – 75%, Kaolin clay – 20.0% and Sphagnum peat - 5.0%
- Maturation of artificial substrate (if any): No
- Moisture: 42% of dry weight
- Presence of macrophytes/animals: Not determined
- Further constituents
- Metals: Not measured
- Sulfides: Not measured
- Organic compounds: Not measured
- Total volatile solids: Not measured
- Acid volatile sulfides: Not measured
- Oil and grease: Not measured
- Petroleum hydrocarbons: Not measured
- Other:
- Eh: Not measured
- Sediment sieved: No
- pH pore water: Not measured
- pH dry matter and/or whole sediment: Adjusted to 7.0 + 0.5 by the addition of calcium carbonate
- Ammonia content of pore water: Not measured
- Total organic carbon (%): 1.7%
- Total inorganic carbon (%): Not measured
- BOD: Not measured
- COD: Not measured
- CEC: Not measured
- Proof of absence of chemical contaminants: Not determined

OTHER TEST CONDITIONS
- Light quality: No data
- Photoperiod: 16 hours light/8 hours dark daily
- Light intensity: 785 – 795 lux

VEHICLE CONTROL PERFORMED: Yes for solvent (acetone) controls

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Justification for using less concentrations than requested by guideline: Not relevant
- Range finding study
- Test concentrations: Not relevant
- Results used to determine the conditions for the definitive study: The definitive final test was performed with concentrations ranging from 62.5 to 1000 mg/kg d.w. to be consistent with the corresponding study on a compositionally similar substance Distilled Grade

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

500 mg/kg sediment dw

Nominal / measured:

nominal

Basis for effect:

emergence rate

Details on results:

- Mortality of test animals at the end of exposure period: The chironomids that did not emerge during the test are considered to have died.

- Total mass of test animals at beginning of test: Not measured

- Changes in body weight of live adults (% of initial weight) at end of exposure period: Not measured

- No. of offspring produced: Not measured

- No. of emerged male and female midges (per vessel and per day): There was no statistically significant effect on the sex ratio of the emerged chironomids. Therefore, pooled data of male and female chironomids were used for further statistical calculations (see Table 3).

- No. of pupae failing to emerge (per vessel and per day): No (dead) pupae and no egg masses were observed at the end of the test (see Table 3).

- Percent emergence per replicate and test concentration: At the end of the test, 95.1% of the midges emerged in the pooled control. There was no statistically significant difference in emergence between the pooled control and the nominal test concentrations up to and including 500 mg/kg d.w. (where there were emergence rates of 90.0 to 97.5%). At the nominal concentration of 1000 mg/kg d.w. a statistically significant reduction between emergence in the pooled control and the test concentration (86.3%) was observed (see Table 3).

- Mean development rate of fully emerged midges (per replicate and treatment rate; male and female midges pooled): There was no statistically significant difference in development rate of chironomids between the blank-control and the solvent-control and the control groups were pooled for further statistical evaluations. The development rate in the pooled control was 0.058/day. There was no significant statistical effect compared to the pooled control in any of the test concentrations (see Table 3).

- Mean individual dry weight of larvae (per larvae and per instar): Not measured

- Number of living worms per replicate: Not measured

- Number of dead worms per replicate: Not measured

- Total biomass per replicate: Not measured

- Morphological abnormalities: Not measured

- Behavioural abnormalities: No behavioural differences in test substance exposed larvae compared to the controls were recorded.

- Other biological observations: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: Groups of chironomids were exposed for a period of 28 days to K2Cr2O7 (Potassium dichromate) concentrations of 5.6, 10, 18, 32 and 56 mg/L and to a control
- Limit test: No
- Dose-response test: Yes
- ECx: The 28-day EC50 for emergence ratio was 21 mg/L (95% Confidence Limits = 16 to 28 mg/L) whilst the 28-day EC50 for development rate was 27 mg/L (95% Confidence Limits = 20 to 35 mg/L)
- Other: The result of the reference substance study was in the same order of magnitude as the earlier studies

Reported statistics and error estimates:

Differences in sex ratio at the end of the test were determined with the Chi2 rx2 – Contingency Table.
Differences of emergence ratio between the blank-control and the solvent-control were determined with the Chi²-2 x 2 Test.
Differences of development rate between the blank-control and the solvent-control were determined with the Student-t test.
All calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Table 1          Summary of the analytical results measured in the OECD TG218 Sediment-Water Toxicity Test on Day 0

 

Form of cardanol	Nominal sediment concentration (mg/kg d.w.)	Sediment concentration (mg/kg d.w.)	Concentration in other samples		Mass balance at Day 28 (mg)
			Pore water (mg/L)	Overlying water (µg/L)
Triene (m/z 297)	62.5	48.8	0.0597	11.1	2.93
	125	94.9	0.112	11.3	5.70
	250	231	0.208	34.0	13.9
	500	479	0.683	59.7	28.7
	1000	961	1.58	211	57.7
Monoene (m/z 301)	62.5	49.5	0.0696	14.3	2.97
	125	97.6	0.106	14.2	5.86
	250	243	0.156	44.2	14.6
	500	489	0.563	71.3	29.4
	1000	969	1.26	204	58.2

Table 2          Summary of the analytical results measured in the OECD TG218 Sediment-Water Toxicity Test on Day 28

 

Form of cardanol	Nominal sediment concentration (mg/kg d.w.)	Sediment concentration (mg/kg d.w.)	Concentration in other samples		Mass balance at Day 28 (mg)
			Pore water (mg/L)	Overlying water (µg/L)
Triene (m/z 297)	62.5	35.1	0.029	0.48	2.11
	125	77.9	0.0796	0.48	4.67
	250	141	0.155	Not detected	8.46
	500	366	0.426	0.74	22.0
	1000	591	0.640	0.86	35.5
Monoene (m/z 301)	62.5	37.1	0.0320	0.51	2.23
	125	78.1	0.0752	0.54	4.69
	250	172	0.136	0.61	10.3
	500	367	0.337	0.91	22.0
	1000	726	0.624	1.0	43.5

 

 Table 3        Summary of the results for the endpoints measured in the OECD TG218 Sediment-Water Toxicity Test

 

Nominal sediment concentration (mg/kg d.w.)	Midges emerged (%) - Emergence ratio (ER) x 100	Development rate (DR) after 28 days
		Mean (day^-1)	% change compared to pooled control
Blank control	96.3	0.058	-
Solvent control	93.8	0.058	-
Pooled control	95.1	0.058	-
6.25	97.5	0.057	-1.0
125	95.0	0.059	+1.3
250	92.7	0.058	+0.7
500	90.0	0.060	+3.4
1000	86.3	0.059	+2.3

 

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study with Chironomus riparius no statistical significant reduction of emergence, compared to the controls, was observed at 500 mg/kg d.w., which corresponds to 419 and 424 mg/kg d.w. based on analysis of cardanol triene (m/z 297) and cardanol monoene (m/z 301), respectively. This resulted in a No Observed Effect Concentration for the emergence ratio endpoint ((NOECER) of 500 mg/kg d.w.

No statistical significant effect on the development rate was observed at 1000 mg/kg d.w. which corresponds to 754 and 839 mg/kg d.w. based on analysis of cardanol-triene (m/z 297) and cardanol monoene (m/z 301), respectively. This resulted in a No Observed Effect Concentration for the development rate endpoint (NOECDR) of 1000 mg/kg d.w.

The 28-day EC50 values for the emergence ratio and development rate endpoints were higher than the highest nominal sediment concentration of 1000 mg/kg d.w., which corresponds to 754 and 839 mg/kg d.w. based on analysis of cardanol-triene (m/z 297) and cardanol monoene (m/z 301), respectively.

Executive summary:

The objective of the study was to assess the effects of prolonged exposure of the sediment-dwelling larvae Chironomus riparius to sediment spiked with Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade). Measured endpoints were the total number of adults emerged (emergence ratio) and the development rate. The study procedure was based on OECD Guideline No. 218 (2004).

 

The batch of Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade) tested, which is a UVCB, was a straw coloured to dark brown liquid. No correction of the data was made for the purity/composition of the test substance.

 

A definitive test was performed with nominal sediment concentrations ranging from 62.5 to 1000 mg/kg dry weight (d.w.), with the concentration series increasing by a factor 2. Furthermore, a blank-control and solvent (acetone)-control were included. Preparation of sediment-water systems started with coating sand with the test substance and the preparation of spiked sediment. Thereafter, test water was added to the sediment. The solvent-control was treated in the same way as the test concentrations. In total 80 chironomids per group (20 per replicate/4 replicates) were exposed to the controls and the test concentrations for a period of 28 days. On Day 0 and Day 28 samples for analysis were taken from overlying water, pore water and sediment. The distribution of Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) within the overlying water, pore water and sediment was determined. Analysis of the samples were performed on two forms of cardanol, i.e. cardanol-triene (m/z 297) and cardanol-monoene (m/z 301), which are the major constituents of the test substance.

 

At the start of the test the test substance concentration in the sediment was between 76% and 98% of the nominal test concentration. During the test, the percentage of the nominal concentrations decreased. The test substance concentration in the sediment was between 56% and 73% of the nominal test concentration on Day 28. Since measured test substance concentrations, based on the different forms of cardanol, in sediment were not in agreement with nominal concentrations, geometrical means were calculated from the measured exposure concentrations at the start and end of the test.

 

The test substance concentrations were < 0.2 mg/L in overlying water and < 1.6 mg/L in pore water for all the different forms of cardanol. The total measured test substance concentration, analysed on the two forms of cardanol, was between 76% and 98% of the nominal initial concentration at Day 0. At the end of the test, the mass balance decreased. The total measured test substance concentration in all exposure phases was between 56% and 73% of the initial levels at Day 28.

 

In both the controls and at all the test concentrations, the first midges emerged on day 14 or 15. There was no statistically significant effect on sex ratio of the emerged chironomids and no statistically significant difference in emergence of chironomids between the blank-control and the solvent-control. Therefore, statistical calculations were performed on pooled data of male and female chironomids and pooled controls. At the end of the test, 95.1% of the midges emerged in the pooled control. There was no statistically significant difference in emergence between that in the pooled control and the nominal test concentrations up to and including 500 mg/kg d.w (emergence in the range 90.0- 97.5%). At the nominal concentration of 1000 mg/kg d.w., a statistically significant difference on emergence between the pooled control and the test concentration was observed (emergence rate of 86.3%). 

 

No (dead) pupae and no egg masses were observed at the end of the test. No behavioural differences in test substance exposed larvae compared to the controls were recorded. The development rate in the pooled control was 0.058/day. There was no significant statistical effect compared to the pooled control in any of the test concentrations.

 

Except for deviations in the temperature, which were considered to have no effect on the test, all acceptability criteria were met and, this study was considered valid.

 

In conclusion, under the conditions of the present study with Chironomus riparius no statistical significant reduction of emergence was observed at nominal concentration of 500 mg/kg d.w. and no statistical significant effect on development rate was observed at a nominal concentration of 1000 mg/kg d.w. This resulted in a No Observed Effect Concentration for the emergence ratio endpoint (NOECER) of 500 mg/kg d.w. and a NOEC for the development rate (NOECDR) of 1000 mg/kg d.w. based on nominal concentrations.

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater sediment:

500 mg/kg sediment dw

Additional information

An OECD guideline 218 study was carried out on to assess the effects of prolonged exposure of sediment-dwelling larvae Chironomus riparius to Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade).

This resulted in a No Observed Effect Concentration for the emergence ratio endpoint (NOECER) of 500 mg/kg d.w. and a NOEC for the development rate (NOECDR) of 1000 mg/kg d.w. based on nominal concentrations.