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

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21st September 2018 to 14th November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Version / remarks:

Study was designed to comply with OECD TG305 dated October 2012
Aqueous and Dietary Exposure

Deviations:

yes

Remarks:

Due to the variability in cardanol concentrations in fish and that most concentrations were below the LOD, there was no depuration phase. The study was terminated after the concentration analysis data for samples taken on day 28 became available.

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms
Tissue samples were collected on Days 1, 3, 7, 14, 21 and 28 of the uptake phase. At each tissue sampling interval the fish were sampled with a small fishing net, rinsed quickly with untreated water, blotted dry and then instantly killed. Subsequently, fish were weighed and then measured for total length.

The number of replicate fish used on each sampling occasions was 2 for the control whilst 4 fish were used for each of the exposure groups.

Each replicate was homogenized (1:1) in Milli-RO water using a blender (Turrax). Two sub-samples of 1 g homogenate each (containing approximately 0.5 g fish each) were taken and used for concentration analysis. The remaining volume was used for lipid extraction.

- Sampling intervals/frequency for test medium samples
Water samples were collected on Day -1 (pre-test) and on uptake Days 0, 1, 3, 7, 14, 21 and 28 of the uptake phase.

Samples were taken from the control and both concentrations and analysed before introduction of the fish to check if the actual test concentrations were in agreement with the expected concentrations.

During the test solutions were sampled by siphoning through inert tubing from a central point in the test chamber. Two 100 ml samples for the control and both test concentrations were taken on each of the sampling occasions.

- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods)
The water and fish tissue samples were analysed using a Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) method. In the method the sample mixture was first separated by liquid chromatography before being ionised and characterised by mass-to-charge ratio and relative abundances using two mass spectrometers in series.

Vehicle:

yes

Details on preparation of test solutions, spiked fish food or sediment:

Stock solutions of Cashew Nutshell Extract, Decarboxylated, Distilled (Distilled Grade) were prepared in dimethylformamide (DMF). The concentrations in the stock solutions were a factor of 10,000 higher than the test concentrations in water, i.e. concentrations in the stock solutions were 3 and 30 mg/l.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

Male and female
TEST ORGANISM
- Common name: Carp
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Age at study initiation (mean and range, SD): Juvenile
- Length at study initiation (length definition, mean, range and SD): The mean total length of the fish was 8.7 ± 0.4 cm, based on measurement of all fish used for the test.
- Weight at study initiation (mean and range, SD): The average wet weight (blotted dry) was 8.99 ± 1.01 g, based on weighing of all fish used for the test.
- Length and weight at termination (mean and range, SD): The mean total length at test termination was 9.9 ± 0.60 cm with a range of 9.0 to 10.8 cm and the average wet weight (blotted dry) was 12.64 ± 2.2 g with a range of 9.62 to 16.12 g.
- Description of housing/holding area:
- Health status: All fish were observed daily to evaluate the number of mortalities and the number of individuals exhibiting signs of abnormal behaviour.
- Feeding during test: Pelleted fish food (of 10% fat and 45% protein content) was supplied at a ration of 2 % of body weight per day. A recalculation of the feeding rate was performed after each sampling point.

ACCLIMATION
- Acclimation period: The carp were maintained in adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands). During the 12 day period prior to the test organisms were kept within the optimum limits for the tested fish species
- Type and amount of food: During the holding period fish were fed daily with pelleted fish food.
- Health during acclimation (any mortalities observed); During the seven days prior to the start of the test mortality was less than 5% and they showed no signs of disease or stress.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: To provide definitive bioconcentration data from a flow-through test that can be directly compared to the relevant regulatory threshold.

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

0 d

Hardness:

The test solutions of the different exposure concentrations had a hardness of 161 to 179 mg CaCO3 per litre.

Test temperature:

The test solutions of the different exposure concentrations had a temperature range of 21.9 to 23.4°C, with a variation less than ± 2°C.

pH:

The test solutions of the different exposure concentrations had a pH between 7.3 and 8.0, with a variation of not more than ± 0.5 units.

Dissolved oxygen:

The test solutions of the different exposure concentrations had a dissolved oxygen level of 6.4 to 8.8 mg/l, which is >60% of saturation required.

TOC:

The adjusted ISO test medium had a Total Organic Carbon (TOC) level of preferably < 0.1 mg C per litre and the control solution had a TOC between 45 and 61 mg/l.

Details on test conditions:

TEST SYSTEM
- Test vessel:
64 litres covered by a removable Perspex plate

- Type (delete if not applicable): open

- Material, size, headspace, fill volume:
Stainless steel, 40 x 40 x 40 cm

- Aeration:
The test solutions were aerated continuously
- Type of flow-through (e.g. peristaltic or proportional diluter):
The stock solutions were dosed via a computer controlled system consisting of microdispensers (Gilson). Via this system the dosed volume entered a mixing flask separately from the medium supply. The medium was supplied via a flow meter. The flow rate was 16 L/h and in the mixing flask the dosed volume and the medium were mixed under continuous stirring at a ratio of 1 to 10,000. The whole system was checked daily.

- Renewal rate of test solution (frequency/flow rate):
The flow-through system allowed for approximately six volume replacements (64 litres) through each aquarium each day. Uneaten food and fecal debris were siphoned daily from the test chambers about an hour after feeding.

- No. of organisms per vessel:
23 in controls and 44 in each of the exposure groups.

- No. of vessels per concentration (replicates):
1
- No. of vessels per control / vehicle control (replicates):
1

- Biomass loading rate:
The loading rate was between 0.1 and 1.0 g fish/litre/day

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
The water used for holding and testing was adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands).

No data is available on parameters related to particulate matter

Dissolved oxygen content and pH were measured three times a week during the uptake phase in each vessel. Temperature was measured continuously in each vessel starting from one day before the introduction of the fish. Hardness was measured at the start and the end of the uptake phase in each vessel.

There were approximately six volume replacements (64 litres) through each aquarium each day in the flow-through system.

OTHER TEST CONDITIONS
- Adjustment of pH:
no data available
- Photoperiod:
A daily photoperiod of 16 hours light/8 hours dark was used.
- Light intensity:
no data available

RANGE-FINDING / PRELIMINARY STUDY
- Other justification for choice of test concentrations: No range finding/preliminary study was conducted so no results are available.

The selection of the test concentrations used in the study was based on the requirement of Test Guideline 305 to be “below its chronic effect level or 1% of its acute asymptotic LC50, within an environmentally relevant range and at least an order of magnitude above its limit of quantification in water by the analytical method used”.

An acute (96-hour) fish lethality test carried out on another member of the category (group), specifically Technical Grade, using Water Accommodated Fractions found that there were no lethal effects at the water solubility limit (0.2 mg/l). Based on this data and the water solubility limit of 0.3 mg/l (300 µg/l) for Distilled Grade the upper test concentration for the Bioaccumulation in Fish study was selected to be 3 µg/l using the 1% adjustment factor. A further test concentration of 0.3 µg/l was also included in the study based on the requirement of the Test Guideline that “If a second concentration is used, it should differ from the one above by a factor of ten”.

Nominal and measured concentrations:

Measured (average over 0 to 28 days):
0.066 µg/l at 0.3 µg/l (nominal) and 0.53 µg/l at 3.0 µg/l (nominal) for cardanol triene (m/z 297)
0.10 µg/l at 0.3 µg/l (nominal) and 0.75 µg/l at 3.0 µg/l (nominal) for cardanol diene (m/z 299)
0.13 µg/l at 0.3 µg/l (nominal) and 1.00 µg/l at 3.0 µg/l (nominal) for cardanol monoene (m/z 301)
0.20 µg/l at 0.3 µg/l (nominal) and 1.20 µg/l at 3.0 µg/l (nominal) for cardanol saturated side (m/z 303)

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured/calculated logPow: 6.2 from an OECD TG117 Partition coefficient (n-octanol-water), HPLC Method study

BASIS FOR CALCULATION OF BCF
The BCF values were calculated from the raw data using in-house estimation software.

Evaluation of the results started with obtaining the uptake curve by plotting the measured concentrations in/on fish against time on arithmetic scales. The curve did not reach a 'plateau'. Therefore, no steady state bioconcentration factor, BCFss, could be calculated. Instead, the individual BCF at each sampling point was calculated, using the formula below:

BCF= Cf/Cw

where: Cf, = concentration in fish
Cw = concentration in water

Normalisation for lipid content was performed for each individual BCF. The concentration in the fish was corrected for the lipid content at each sampling point, using the formula below:

CfL = (0.05Cf)/L


where: Cf,L = lipid-normalized concentration in fish
L = lipid fraction
Cf = concentration of test substance in fish

In the study the extent of the accumulation of the different forms of cardanol (i.e. triene, diene, monoene and saturated side chain) was generally low with many fish not showing measured levels above the limit of detection of the analytical method for fish tissues. This was despite the fish being exposed to detectable aqueous levels of the different forms of cardanol. Bioconcentration factors (BCFs) were derived for each sampling occasion during the uptake phase (i.e. days 1, 3, 7, 14, 21 and 28). However, no plateau was evident for any of the BCFs for any forms of cardanol and since no steady state concentrations were achieved the BCFs at steady state (BCFss) could not be determined.

The uptake rate constant (k1) is the numerical value defining the rate of increase in the concentration of test substance in/on test fish (or specified tissues thereof) when the fish are exposed to that chemical. However, these constants for the different forms of cardanol could not be calculated in the study given the nature of the patterns of uptake observed for the different forms of cardanol.

Lipid content:

>= 3 - < 4 %

Time point:

end of exposure

Remarks on result:

other: Whole body samples

Key result

Conc. / dose:

< 0.3 µg/L

Type:

other: BCF for cardanol triene (m/z 297)

Value:

< 100 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Calculated using the day 28 data

Remarks on result:

other: Conc. in environment / dose: 0.066 µg/l at 0.3 µg/l (nominal) and 0.53 µg/l at 3.0 µg/l (nominal). The low level of uptake of the test substance over the 28 day exposure period meant that no plateau was observed.

Key result

Conc. / dose:

ca. 3 µg/L

Type:

other: BCF for cardanol triene (m/z 297)

Value:

ca. 397 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Calculated using the day 28 data

Remarks on result:

other: In cases of all forms of cardanol. The low level of uptake of the test substance over the 28 day exposure period meant that no plateau was observed.

Conc. / dose:

ca. 0.3 µg/L

Type:

other: BCF for cardanol diene (m/z 299)

Value:

ca. 313 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Calculated using the day 28 data

Conc. / dose:

ca. 3 µg/L

Type:

other: BCF for cardanol diene (m/z 299)

Value:

ca. 326 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Calculated using the day 28 data

Conc. / dose:

ca. 0.3 µg/L

Type:

other: BCF for cardanol monoene (m/z 301)

Value:

ca. 266 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Calculated using the day 28 data

Conc. / dose:

ca. 3 µg/L

Type:

other: BCF for cardanol monoene (m/z 301)

Value:

ca. 182 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Calculated using the day 28 data

Conc. / dose:

ca. 0.3 µg/L

Type:

other: BCF for cardanol saturated side chain (m/z 303)

Value:

< 100 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Calculated using the day 28 data

Conc. / dose:

<= 3 µg/L

Type:

other: BCF for cardanol saturated side chain (m/z 303)

Value:

ca. 100 L/kg

Basis:

whole body w.w.

Calculation basis:

other: Calculated using the day 28 data

Key result

Elimination:

no

Parameter:

other: Due to the variability in the measured cardanol concentrations in fish and the fact that most concentrations were below the LOD, no depuration phase was performed

Remarks on result:

not determinable because of methodological limitations

Validity criteria fulfilled:

yes

Conclusions:

Bioconcentration factors (BCFs) were calculated for the four forms of cardanol, the triene (m/z 297), diene (m/z 299, monoene (m/z 301) and saturated side chain (m/z 303) using groups of carp exposed to concentrations of 0.3 and 3.0 µg/l over a 28 day uptake phase.

BCF values of < 100 L/kg in all cases for the 0.3 µg/l nominal exposure group and <100 to 397 L/kg for the 3.0 µg/l nominal exposure group were calculated for cardanol triene where the Limit of Detection was 100 L/kg.

For cardanol diene the calculated BCFs were < 100 to 619 L/kg for the 0.3 µg/l nominal exposure group and <100 to 326 L/kg for the 3.0 µg/l nominal exposure group.

BCFs of < 100 to 882 L/kg for the 0.3 µg/l nominal exposure group and <100 to 182 L/kg for the 3.0 µg/l nominal exposure group were calculated for cardanol monoene.

For cardanol saturated side chain the calculated BCFs were< 100 L/kg in all cases for both the 0.3 µg/l and 3.0 µg/l nominal exposure groups.

The low Bioconcentration Factors (<900 L/kg)) for the four different forms of cardanol (monoene, diene, triene and saturated side chain) in the UVCB measured in the aqueous exposure study on Distilled Grade are also consistent with the results obtained from a corresponding dietary study on the other source substance Distillation Residue Grade.

For Distillation Residue Grade dietary Biomagnification Factors (BMFs) and overall depuration rate (k2) constants were derived for the three forms of cardanol measured. Bioconcentration Factors (BCFs) were also derived using the data generated in the study. The tentative BCF values (L/kg) derived from the study (based on the methods of Brooke and Crookes, 2012 and Inoue et al 2012) were 837-893, 414-478 and 280-362 for the monoene, diene and triene forms of cardanol respectively. These values are significantly below the 2000 L/Kg threshold for a substance to be considered to be bioaccumulative. The estimated BCF values (L/kg) for the different forms of cardanol based on the k2 values (and the Brooke and Crookes, 2012 method) are 672, 333 and 245 for the monoene, diene and triene forms of cardanol respectively. These results also indicate that the different forms of cardanol (monoene, diene and triene) did not markedly bioaccumulate and were significantly below the 2000 L/Kg threshold. As a result the test substance Distillation Residue Grade is not considered to be bioaccumulative. The estimated BCF values for the different forms of cardanol in the dietary study are consistent with those from the Distilled Grade study carried out via the aqueous exposure route.

Executive summary:

Bioconcentration factors (BCFs) were calculated for the four forms of cardanol, the triene (m/z297), diene (m/z299, monoene (m/z301) and saturated side chain (m/z303) using groups of carp exposed to concentrations of 0.3 and 3.0 µg/l over a 28 day uptake phase according to OECD Guideline 305 (Bioaccumulation in Fish study).

Stock solutions were prepared in dimethylformamide (DMF) and dosed via a computer-controlled system consisting of micro-dispensers into a mixing flask separately from the dilution medium supply (ISO medium). Medium was supplied via a flow meter at a rate of 16 L/h. In the mixing flask, the dosed stock solution volume and the dilution medium were mixed at a ratio of 1 : 10,000 with continuous stirring.

At the start of the exposure period, 44 fish were exposed to each target concentration and 23 fish were exposed to a DMF control. Samples were taken from the test medium and from the fish during the exposure period.

Variation was observed between duplicate samples taken from the test solutions and also between time points. No steady water concentration was maintained for any of the four cardanol components at either target concentration of the UVCB, i.e. the measured concentrations varied outside the ± 20% window.

The measured concentrations in most fish samples were below the limit of detection (LOD) of the analytical method (i.e. <30 µg/kg). In case of concentrations in fish being below the LOD, the BCF was indicated to be <100 L/kg. All other, calculated, BCFs are indicative because the measured concentrations in fish were determined by extrapolation.

Since there was no steady-state concentration, the BCFss could not be determined. The individual BCFs, although indicative, showed that accumulation in fish was always below 900 L/kg. BCF values of < 100 L/kg in all cases for the 0.3 µg/l nominal exposure group and <100 to 397 L/kg for the 3.0 µg/l nominal exposure group were calculated for cardanol triene. For cardanol diene the calculated BCFs were < 100 to 619 L/kg for the 0.3 µg/l nominal exposure group and <100 to 326 L/kg for the 3.0 µg/l nominal exposure group. BCFs of < 100 to 882 L/kg for the 0.3 µg/l nominal exposure group and <100 to 182 L/kg for the 3.0 µg/l nominal exposure group were calculated for cardanol monoene. For cardanol saturated side chain the calculated BCFs were< 100 L/kg in all cases for both the 0.3 µg/l and 3.0 µg/l nominal exposure groups.

Due to the variability in the measured cardanol concentrations in fish and the fact that most concentrations were below the LOD, no depuration phase was performed.

When corrected for lipid concentrations in fish, the BCF values were slightly higher than without lipid correction. However, the overall results were similar.

A test item is considered to be bioaccumulative when the BCF is >2000 L/kg. The obtained individual BCF values for the four cardanol components in the UVCB, both before and after lipid normalisation, were <2000 L/kg.

The low Bioconcentration Factors (<900 L/kg)) for the four different forms of cardanol (monoene, diene, triene and saturated side chain) in the UVCB measured in the aqueous exposure study on Distilled Grade are also consistent with the results obtained from a corresponding dietary study on the other source substance Distillation Residue Grade,

For Distillation Residue Grade dietary Biomagnification Factors (BMFs) and overall depuration rate (k₂) constants were derived for the three forms of cardanol measured. Bioconcentration Factors (BCFs) were also derived using the data generated in the study. The tentative BCF values (L/kg) derived from the study (based on the methods of Brooke and Crookes, 2012 and Inoue et al 2012) were 837-893, 414-478 and 280-362 for the monoene, diene and triene forms of cardanol respectively. These values are significantly below the 2000 L/Kg threshold for a substance to be considered to be bioaccumulative. The estimated BCF values (L/kg) for the different forms of cardanol based on the k₂ values (and the Brooke and Crookes, 2012 method) are 672, 333 and 245 for the monoene, diene and triene forms of cardanol respectively. These results also indicate that the different forms of cardanol (monoene, diene and triene) did not markedly bioaccumulate and were significantly below the 2000 L/Kg threshold. As a result the test substance Distillation Residue Grade is not considered to be bioaccumulative. The estimated BCF values for the different forms of cardanol in the dietary study are consistent with those from the Distilled Grade study carried out via the aqueous exposure route.

Description of key information

Data from an OECD TG305 Bioaccumulation in Fish study carried out to GLP and using carp showed that there was no uptake of the four forms of cardanol (monoene, diene, triene and saturated side) measured in the study. These low molecular weight constituents represent 78% of the test substance and are considered to have the potential to bioaccumulate based on high octanol-water partition coefficients. However, no uptake was observed in the study and the individual BCF values obtained for the four forms of cardanol were <2000 L/kg, both before and after lipid normalisation. Distilled Grade, therefore, is not considered to be bioaccumulative.

Key value for chemical safety assessment

BCF (aquatic species):

882 L/kg ww

Additional information

The substance consists of multiple components with BCFs as listed below:

Cardanol monoene:                        <100 to 882 L/kg

Cardanol diene:                                <100 to 619 L/kg

Cardanol triene:                               <100 to 397 L/kg

Cardanol saturated side-chain:  <100 L/kg in both exposure concentrations

All the BCFs indicate a low potential for bioaccumulation. For the purposes of the chemical safety assessment the highest experimental figure for the BCF of 882 L/kg has been used as a worst case.