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Environmental fate & pathways

Bioaccumulation: aquatic / sediment

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Reference
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15th December 2020 to 20th January 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Conducted under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -III: Dietary Exposure Bioaccumulation Fish Test
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Manufacturer
Batch (Lot) Number: FP-0306
Physical Description: Straw coloured to dark brown liquid
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%

- Purity, including information on contaminants, isomers, etc.: 100% (UVCB)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated at approximately 4˚C in the dark
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stable
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Stable
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Stable
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: Samples of stock fish taken on Day 0 and samples of test fish taken on Day 14 of uptake phase and on Days 1, 4, 7, 14 and 21 of depuration phase.
- Sampling intervals/frequency for test medium samples: Diet was analyzed on Days 0, 6, 7 and 13 of uptake phase.
- Sample storage conditions before analysis: Samples analyzed on the day of sampling or stored frozen prior to analysis.
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Test Samples – Diet Analysis
The test samples were analyzed on the day of sampling, or stored frozen prior to analysis. A mass (5 g) of fish food was weighed into a centrifuge tube, then an aliquot of water (20 mL) and an aliquot of acetonitrile (100 mL) were added. The samples were vortex mixed for approximately 30 seconds, then sonicated for 15 minutes. The samples were centrifuged for 15 minutes at 15,000 g. Where necessary, the supernatant was diluted into the calibration range with acetonitrile: water (10:2 v/v). The resulting supernatant was vialled for analysis.

Test Samples – Fish Analysis
The test samples were analyzed on the day of sampling or allowed to equilibrate to room temperature prior to analysis. A whole fish was macerated and approximately 5 g of this fish paste was accurately weighed into a 500 mL centrifuge tube. An aliquot of water (20 mL) and an aliquot of acetonitrile (100 ml) were added. The mixture was macerated with a high speed macerator for at least 1 minute and then vortex mixed for approximately 30 seconds, followed by sonication for 1 minute. The samples were centrifuged for 15 minutes at 15,000 g. The resulting supernatant, where necessary was diluted into the calibration range with an acetonitrile: water mixture (10:2 v/v).
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION OF SPIKED FISH FOOD
- Details on fish food (source, fat content as supplied, etc): Fish were fed with Inicio Plus 1.1 mm trout pellets (56% protein and 18% fat content)
- Details of spiking (e.g. i) liquid test material (neat); ii) with a vehicle (corn or fish oil); or iii) using an organic solvent: Corn Oil used as a vehicle
- Quantity of corn or fish oil vehicle, if used, per unit mass of fish food: 4 mL of corn oil was added to 210 g of trout pellets
- Chemical name of vehicle (organic solvent), if used: None
- Method of mixing: Mixed thoroughly by shaking by hand for one minute (at the start and end of mixing) and on a rotary mixer for approximately 30 minutes to ensure thorough incorporation and distribution.
- Equilibration time: 30 minutes
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Source: An accredited live fish supplier
- Age at study initiation (mean and range, SD): Approximately 33 weeks
- Length at study initiation (length definition, mean, range and SD): Greater than 6 cm
- Weight at study initiation (mean and range, SD): Control fish 8.74g (7.25 – 10.65g) SD = 0.96. Test fish 8.75g (7.22 – 10.62g) SD = 0.93.
- Weight at termination (mean and range, SD): Control fish 23.17g (16.00 – 33.71g) SD = 3.84. Test fish 22.56g (16.30 – 29.04g) SD = 3.58.
- Method of breeding: Natural
- Lipid content at test initiation (mean and range, SD): 4.60% (4.20 – 5.20%) SD = 0.41. Determined from fish frozen on Day 1
- Health status: Good
- Description of housing/holding area: Fibreglass holding tanks with continual single pass water renewal
- Feeding during test
- Food type: Inicio Plus 1.1 mm trout pellets
- Amount: 2% Body weight
- Frequency: Once per day

ACCLIMATION
- Acclimation period: At least 2 weeks
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: Inicio Plus 1.1 mm trout pellets Ad Lib
- Feeding frequency: Once per day
- Health during acclimation (any mortality observed): 0% mortality
Route of exposure:
feed
Justification for method:
dietary exposure method used because stable, measurable water concentrations cannot be maintained
Remarks:
See "Any other information on materials and methods incl. tables" for justification.
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
14 d
Total depuration duration:
21 d
Hardness:
Hardness in the control tank was 122 mg/L as CaCO3, whilst it was 120 mg/L as CaCO3 in the treatment tank.
Test temperature:
Temperature of 13oC was maintained in both tanks throughout the study.
pH:
pH measurements in the control tank ranged from 7.7 to 8.0, while in the treatment tank these ranged from 7.6 to 8.0 (see Table 1).
Dissolved oxygen:
Dissolved oxygen(mg O2/l) measurements in the control tank ranged from 8.3 to 10.5, while in the treatment tank these ranged from 8.5 to 10.5 (see Table 1).
TOC:
TOC in the control tank was 1.1 mg C/L , whilst it was 1.0 mg C/L in the treatment tank.
Salinity:
N/A
Conductivity:
No data
Details on test conditions:
TEST SYSTEM
- Test vessel: Glass test vessels (approximately 140 litres in volume)
- Type (delete if not applicable): Open (covered)
- Material, size, headspace, fill volume: Approximately 100 litres fill volume
- Aeration: Via air stone
- Type of flow-through (e.g. peristaltic or proportional diluter): Metering pump
- Renewal rate of test solution (frequency/flow rate): 800 mL/min
- No. of organisms per vessel: 50
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: Control tank: 4.37 g/L ,Test tank: 4.38 g/L (per 100L) – 0.38 g/L (1152 L water renewal per day)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The water used for holding and testing was dechlorinated by passage through an activated carbon filter (Fleck 2750 Duplex Dechlorination Unit) and a proportion of the incoming water was softened (Elga Nimbus 1248D Duplex water softener) and then remixed with the main supply to provide a water hardness of approximately 140 mg/L as CaCO3 (within the range 110 to 160 mg/L as CaCO3) and pH 6.0 to 8.5. After dechlorination and softening the water was then passed through a series of computer controlled heat exchangers to achieve the required temperature. When a "single pass" system was not possible, the water was recirculated via a suitable purification filter.
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio: No data
- Conductance: No data
- Holding medium different from test medium: No
- Intervals of water quality measurement: Daily
- Intervals of test medium replacement: 11 times per day

OTHER TEST CONDITIONS
- Adjustment of pH: Not required
- Photoperiod: 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods
- Light intensity: 741 – 809 Lux
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall daily feeding rate used in the study: 2% Body weight
- For OECD 305 part III (dietary exposure fish bioaccumulation), number of feeds per day (number of feeds daily ration split between): Once daily
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food before test start taking into account the contribution from the corn or fish oil vehicle, if used: 18% Diet plus 3% Corn Oil (21%)
- For OECD 305 part III (dietary exposure fish bioaccumulation), overall lipid content of spiked food after end of exposure taking into account the contribution from the corn or fish oil vehicle, if used: 18% Diet plus 3% Corn Oil (21%)
Nominal and measured concentrations:
The nominal concentrations of the different forms of cardanol in the spiked diet were consistent with the measured data throughout the study (see Table 2). In addition, analysis of triplicate samples confirmed that that the different forms of cardanol were homogeneous in the spiked diet.
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Measured/calculated logPow: Measured logPow of >6.2
- Results from toxicokinetic study: No data
- Results from residue study: No data
- Monitoring data: No data

BASIS FOR CALCULATION OF BCF
- Estimation software: BCF and BMF values were estimated using kinetic analysis
- Result based on measured log Pow of: 6.2 for Distillation Residue Grade
- Result based on calculated log Pow of: 8.72 for cardanol monoene, 8.51 for cardanol diene and 8.37 for cardanol triene
Lipid content:
4.4 %
Time point:
end of exposure
Lipid content:
4.6 %
Time point:
start of exposure
Key result
Conc. / dose:
1 000 µg/g food
Temp.:
13 °C
pH:
7.8
Type:
BMF
Value:
0.013 dimensionless
Basis:
whole body w.w.
Time of plateau:
14 d
Calculation basis:
other: kinetic, lipid-corrected, growth-corrected
Remarks on result:
other: Value is a lipid-corrected growth-corrected kinetic BMF
Remarks:
Key result for cardanol triene
Key result
Conc. / dose:
1 000 µg/g food
Temp.:
13 °C
pH:
7.8
Type:
BMF
Value:
0.018 dimensionless
Basis:
whole body w.w.
Time of plateau:
14 d
Calculation basis:
other: kinetic, lipid-corrected, growth-corrected
Remarks on result:
other: Value is a lipid-corrected growth-corrected kinetic BMF
Remarks:
Key result for cardanol diene
Key result
Conc. / dose:
1 000 µg/g food
Temp.:
13 °C
pH:
7.8
Type:
BMF
Value:
0.039 dimensionless
Basis:
whole body w.w.
Time of plateau:
14 d
Calculation basis:
other: kinetic, lipid-corrected, growth-corrected
Remarks on result:
other: Value is a lipid-corrected growth-corrected kinetic BMF
Remarks:
Key result for cardanol monoene
Key result
Elimination:
yes
Parameter:
other: t½g
Remarks:
(cardanol triene)
Depuration time (DT):
0.676 d
Remarks on result:
other: Value is the growth corrected half life
Remarks:
Key result for cardanol triene
Key result
Elimination:
yes
Parameter:
other: t½g
Remarks:
(cardanol diene)
Depuration time (DT):
0.929 d
Remarks on result:
other: Value is the growth corrected half life
Remarks:
Key result for cardanol diene
Key result
Elimination:
yes
Parameter:
other: t½g
Remarks:
(cardanol monoene)
Depuration time (DT):
1.653 d
Remarks on result:
other: Value is the growth corrected half life
Remarks:
Key result for cardanol monoene
Key result
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
1.025
Remarks on result:
other: Key result for cardanol triene
Key result
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
0.746
Remarks on result:
other: Key result for cardanol diene
Key result
Rate constant:
growth-corrected depuration rate constant (d-1)
Value:
0.419
Remarks on result:
other: Key result for cardanol monoene
Details on kinetic parameters:
- Uptake rate constant k(s): The calculated uptake rate constants from food (kf) were 0.0040, 0.0033 and 0.0033 day-1 for the monoene, diene and triene forms of cardanol
- Depuration rate constant k(e): The calculated overall depuration rate constants (k2) were 0.428, 0.755 and 1.034 day-1 for the monoene, diene and triene forms of cardanol
- Indication of bi- or multiphasic kinetics: Biphasic
- Computation / data analysis: Concentration-time data in whole fish were analyzed using the software R version 4.0.2 (2020-06-22) (The R Foundation for Statistical Computing) and OECD-TG305 R-Package bcmfR (v0.4-18) consistent with OECD 305 Guidance (2017). Model fitting and simulations were performed using lipid food contents of 18%. Three optimization models were tested against the data for the Cardanol monoene, diene, and triene constituents of Cashew Nutshell Extract, Decarboxylated, Distillation Residue (Distillation Residue Grade), namely linear regression, a Box-Cox transformation and a Natural Log transformation. For all the forms of cardanol optimum results were obtained using a normal linear regression fit.
Metabolites:
Not determined
Results with reference substance (positive control):
N/A
Details on results:
Mortality of test organisms: No mortalities recorded
- Behavioural abnormalities: None observed
- Observations on feeding behavior: Normal feeding behavior observed
- Observations on body length and weight: Acceptable for test species
- Reproduction during test period: Not relevant
- Other biological observations: None
- Organ specific bioaccumulation: No data
- Bound residues forming a plateau: No data
- Mortality and/or behavioural abnormalities of control: None
- Loss of test substance during test period: None
- Non-eliminated residues (NER) at the end of elimination phase: None
- Results with vehicle control: Not relevant

OECD TG305 Bioaccumulation in Fish Study – Data Tables


 


Physico-chemical parameters


The pH and dissolved levels measured in the control and treatment tanks satisfied the validity criteria for the test.


Table 1        Water quality (pH and dissolved oxygen levels) in the control and treatment tanks during the dietary study


































 


 


Parameter



 


Range of measurements over different periods in different tanks


 



 


Uptake phase



 


Depuration phase


 



 


Control tank



 


Test tank



 


Control tank



 


Test tank


 



pH



7.7 – 8.0



7.7 – 7.9



7.7 – 8.0



7.6 – 8.0



Dissolved oxygen (mgO2/l)



8.3 – 9.9



8.5 – 10.1



9.1 – 10.5



8.6 – 10.5



 


 


 


Concentration of the test substance (as different forms of cardanol) in the spiked diet


The concentrations of each of the forms of cardanol in the spiked diet were within 20% during the uptake phase and consistent with the nominal spiked level of 1000 µg/g (Table 2). The homogeneity of the test substance in the spiked diet did not vary more than + 15% from the mean.


Table 2        Concentration data for different forms of cardanol in the spiked diet













































 


Test day


 



 


Mean measured concentrations of different forms of cardanol in spiked diet (µg/g)


 



 


Monoene


 



 


Diene



 


Triene



0



938



1090



1061



6



894



1050



1045



7



858



1070



986



13



891



1007



1088



Overall mean



895



1054



1045



 


 


 


Concentration of the test substance (as different forms of cardanol) in the tissues of fish exposed to the spiked diet


The mean measured tissue concentrations of the different forms of cardanol in fish at Day 14 of the uptake phase were 7.46 µg/g, 3.91 µg/g and 2.80 µg/g for the monoene, diene and triene forms respectively (see Table 3). The mean measured concentration of the different forms was <0.10 µg/g (the limit of quantitation) for each of the forms of cardanol at Day 21 of the depuration phase.


 


 


 


 


Table 3        Concentration data for different forms of cardanol in the tissues of fish treated with spiked diet

























































 


Test day


 



 


Mean measured concentrations of different forms of cardanol in fish tissues (µg/g)


 



 


Monoene


 



 


Diene



 


Triene



0



<0.10



<0.10



<0.10



14 (Uptake)



7.46



3.91



2.80



15 (Depuration day 1)



5.45



2.19



1.17



18 (Depuration day 4)



1.62



0.587



0.285



21 (Depuration day 7)



0.705



0.188



0.134



28 (Depuration day 14)



0.231



<0.10



<0.10



35 (Depuration day 21)



<0.10



<0.10



<0.10



 


Lipid content


The lipid content of fish tissue at the start of the study was 4.6 + 0.4 % (range 4.2 to 5.2%). The mean lipid content in the treatment fish (based on measurements made on days 14 and 21 of the depuration phase) was 4.4% indicating that this parameter had not changed markedly during the course of the study.


Growth rate


The fish in the control and treatment groups showed similar levels of growth over the course of the uptake and depuration phases of the study (namely 0.412 g/day for control fish and 0.395 g/day for treatment fish based on mean weights at Days 0 and 35)(see Table 4).


Table 4        Summary of the weight data for control and treatment group animals at the beginning (Day 0) and end (Day 35) of the dietary exposure test































 


Fish group



 


Weight at Day 0 (g)



 


Weight at Day 35


 



 


Mean + SD



 


Range



 


Mean + SD



 


Range


 



 


Control


 



 


8.74 + 0.96



 


7.25 – 10.65



 


23.17 + 3.84



 


16.00 – 33.71



 


Treatment


 



 


8.75 + 0.93



 


7.22 – 10.62



 


22.56 + 3.58



 


16.30 – 29.04



 


Growth rate constants were (kg) were calculated by performing a linear least square correlation on the individual data of the control and treatment groups. The slopes of the linear regression were compared statistically using the Students t-test (p=0.05).


 


Food ingestion rate


The set feed ingestion rate constant (I) was calculated using the desired amount of food to be fed per day, weight of the fish and number of fish remaining in the each tank. Effective feeding rate corrected for growth (Ig) were adjusted based on the weights of sampled fish at each interval during the study.


Assimilation efficiency


The assimilation efficiency (α) is the efficiency of absorption of the test substance across the gut. The equations and parameters used to calculate the assimilation efficiency are given in the OECD TG305 Guideline. The assimilation efficiency for whole fish tissue in the treatment group was 0.348 for cardanol monoene, 0.291 for cardanol diene and 0.286 for cardanol triene.


Calculation of dietary study endpoints relevant to bioaccumulation


Table 5 summarises the data obtained for the endpoints measured for the different forms of cardanol in the dietary exposure study.


During the depuration phase elimination of the accumulated forms of cardanol was rapid and 95% loss had been measured for both the diene and triene forms by day 7 and by day 14 for the monone form (where elimination after 7 days was 84%). All forms of cardanol were below the limit of quantification (<0.10 µg/g) by either depuration day 14 (diene and triene forms) or day 21 (monoene form). The time to reach 50% clearance of the monoene, diene and triene forms of cardanol was 1.653, 0.929 and 0.676 days respectively (see Table 5).


The depuration rate constant (k2) was calculated by plotting the natural logarithm (ln) of measured concentrations of the three forms of cardanol for the depuration phase verses time (day) and calculating a least squares correlation. The slope of the line was reported as the overall depuration constant (k2) and the intercept of the line was reported as the natural logarithm of the derived time zero concentration (C0,d). In the treatment tank the k2 values for the whole fish tissues were 0.428, 0.755 and 1.034 respectively for cardanol monoene, diene and triene. The resulting derived time zero concentrations for cardanol monoene, diene and triene were 8.28 µg/g, 4.64 µg/g and 3.30 µg/g respectively.


The BMFK (kinetic dietary biomagnification factors) values were 0.00927, 0.00440 and 0.00316 for the monoene, diene and triene forms of cardanol. The corresponding BMFKg (growth corrected kinetic dietary biomagnification factors) values were 0.00946, 0.00445 and 0.00318 for the monoene, diene and triene forms of cardanol.


The BMFKgL (lipid corrected growth corrected kinetic dietary biomagnification factors) values were 0.0387, 0.0182 and 0.0130 for the monoene, diene and triene forms of cardanol.


To assess the extent to which the different forms of cardanol present in Distillation Residue Grade exhibit bioaccumulation in fish two approaches have been used, namely:



  • Calculating tentative BCFs using a model predicted uptake rate constant (k1) and the depuration rate constant (k2) determined from the dietary exposure study.

  • Using the depuration rate constant (k2) as a surrogate for a BCF value based on the approach described in Brooke and Crookes (2012), in which the use of k2 as an alternative metric for assessment of the B (BCF >2,000 L/kg) or vB (BCF >5,000 L/kg) properties of a substance.


The tentative BCF values (L/kg) derived from the studies were 837-893, 414-478 and 280-362 for the monoene, diene and triene forms of cardanol respectively (based on the methods of Brooke and Crookes, 2012 and Inoue et al 2012) which are significantly below the 2000 L/Kg threshold for a substance to be considered to be bioaccumulative.


The overall depuration rate (k2) constants derived in the study were 0.428, 0.755 and 1.034 for the monoene, diene and triene forms of cardanol respectively. All these values were higher than the critical < 0.178 (no lipid normalisation) value for k2 which corresponds to the threshold BCF value (no lipid normalisation) of >2000 (Brooke and Crookes, 2012). Using the regression equation between log BCF and log K2 given in Table 3 (for data with no lipid normalisation) the estimated BCF values (L/kg) for the different forms of cardanol are 672, 333 and 245 for the monoene, diene and triene forms of cardanol respectively. These results indicate that the different forms of cardanol (monoene, diene and triene) did not markedly bioaccumulate and the estimated BCFs were significantly below the 2000 L/kg threshold.


 


 


Table 5        Summary of the endpoints measured for the different forms of cardanol in the dietary exposure study

















































































Endpoints



 


Calculated values for different forms of cardanol


 



 


Monene



 


Diene



 


Triene


 



 


Chemical concentration in the food (Cfood) (µg/g)


 



 


1000 (nominal)


895 (Measured)



 


1000 (nominal)


1054 (measured)



 


1000 (nominal)


1045 (measured)



 


Substance assimilation efficiency (α)


 



 


0.348



 


0.291



 


0.286



 


Uptake rate constant from food (kf)


(day-1)


 



 


0.0040



 


0.0033



 


0.0033



 


Overall depuration rate constant (k2)(day-1)


 



 


0.428



 


0.755



 


 


1.034



 


Growth-corrected depuration rate constant (k2g) (day-1)


 



0.419



 


0.746



 


1.025



 


Measured time zero concentration (C0,m)(µg/g)


 



 


7.46 (+ 2.53)



 


3.91 (+ 1.75)



 


2.80 (+ 1.22)



 


Derived time zero concentration of depuration phase (C0,d)(µg/g)


 



 


8.28



 


4.64



 


3.30



 


Growth-corrected half-life (t½)(day)


 



 


1.653


 



 


0.929



 


0.676



 


Kinetic dietary Biomagnification Factor (BMFK)


 



 


0.00927



 


0.0044



 


0.0032



 


Growth corrected kinetic dietary Biomagnification Factor (BMFKgL)


 



 


0.00946



 


0.0045



 


0.0032



 


Lipid corrected growth corrected kinetic dietary Biomagnification Factor (BMFKgL)


 



 


0.0387



 


0.0182



 


0.0130



 

Validity criteria fulfilled:
yes
Conclusions:
A reliable without restrictions (Klimisch Code 1) definitive dietary study has been carried out according to the OECD TG305 method using a total of 50 juvenile rainbow trout in each of the control and treatment tanks. It was planned to use the aqueous exposure route in the study for ease of comparison of the resulting data with that for Distilled Grade, but attempts at two different test laboratories were not successful due to issues with the analysis of the test substance and maintenance of stable, measurable water concentrations. The dietary exposure study incorporated a 14 day uptake phase where test animals were exposed to diet spiked with 1000 mg/kg Distillation Residue Grade followed by a 21 day depuration phase where animals were fed unspiked diet. Fish in the control tank were fed unspiked diet throughout the study. Fish were sampled on day 14 of the uptake phase and on days 1, 4, 7, 14 and 21 of the depuration phase. The concentrations of the three key forms of cardanol present in the test substance (monoene, diene and triene) were measured in the spiked diet and fish tissues.

During the uptake phase levels of the three forms of cardanol increased in the animals receiving the spiked diet. The mean concentrations on day 14 (the measured time zero concentrations) were 7.46 µg/g for the monone form, 3.91 µg/g for the diene form and 2.80 µg/g for the triene form. These constituents of the test substances were not detectable in fish from the control tank which received unspiked diet. During the depuration phase elimination of the accumulated forms of cardanol was rapid and 95% loss had been measured for both the diene and triene forms by day 7 and by day 14 for the monone form (where elimination after 7 days was 84%). All forms of cardanol were below the limit of quantification (<0.10 µg/g) by either depuration day 14 (diene and triene forms) or day 21 (monoene form). The time to reach 50% clearance of the monoene, diene and triene forms of cardanol was 1.653, 0.929 and 0.676 days respectively.

The BMFK (growth corrected kinetic dietary biomagnification factors) values were 0.00946, 0.00445 and 0.00318 for the monoene, diene and triene forms of cardanol. The BMFKgL (lipid corrected growth corrected kinetic dietary biomagnification factors) values were 0.0387, 0.0182 and 0.0130 for the monoene, diene and triene forms of cardanol. The lipid content of the fish determined on Days 14 and 21 of the depuration phase had not changed from that measured at the start of test.

Bioconcentration Factors (BCFs) were also derived using the data generated in the study based on the methods of Brooke and Crookes, 2012 and Inoue et al, 2012). The tentative BCF values (L/kg) derived from the study were 837-893, 414-478 and 280-362 for the monoene, diene and triene forms of cardanol respectively which are significantly below the 2000 L/Kg threshold for a substance to be considered to be bioaccumulative.

On the basis of the data generated in the study the different forms of cardanol and the test substance Distillation Residue Grade are not considered to be bioaccumulative.

References:

Brooke, D and M. Crookes (2012) Depuration rate constant: growth correction and use as an indicator of bioaccumulation potential. Environment Agency Report LIT 7371, Environment Agency Bristol, UK.

Inoue, Y., Hashizume, N., Yoshida, T., Murakami, H., Suzuki, Y., Koga, Y., Takeshige, R., Kikushima, E., Yakata, N. and M. Otsuka (2012) Comparison of bioconcentration and biomagnification factors for poorly water-soluble chemicals using common carp (Cyprinus carpio L.). Archive of Environmental Contamination and Toxicology, 63(2), 241–248.
Executive summary:

A reliable without restrictions (Klimisch Code 1) definitive dietary study has been carried out according to the OECD TG305 method using a total of 50 juvenile rainbow trout (>6 cm in length at test initiation) in each of the control and treatment tanks. It was planned to use the aqueous exposure route in the study for ease of comparison of the resulting data with that for Distilled Grade.  Therefore, conduct of the test on Distillation Residue Grade via the aqueous exposure route has been attempted at two different test laboratories. In the first study it did not prove possible to obtain a sufficiently sensitive and reliable analytical method to quantify the different forms of cardanol (monoene, diene and triene) present in Distillation Residue Grade. In the second study a suitable analytical method was developed and validated. However, water sample analysis showed that it was not possible to maintain stable exposure concentrations of the different forms of cardanol in the Distillation Residue Grade test. Therefore, the study has been carried out using the Dietary Exposure Bioaccumulation in Fish Test Method in OECD TG305.


 


The dietary exposure study incorporated a 14 day uptake phase where test animals were exposed to Inicio Plus 1.1 mm trout pellets Ad Lib) diet spiked with 1000 mg/kg Distillation Residue Grade. This dose had been shown to be palatable to the fish in a previous dose range finding study. At this point the concentrations of the three key forms of cardanol present in the test substance (monoene, diene and triene) were measured in 10 individual fish (see Table 3). Ten fish from the control tank which had been exposed to unspiked diet over the 14 day period were also analysed. The uptake phase was followed by a 21 day depuration phase during which period the accumulated concentrations of the three forms of cardanol measured at the end of the uptake phase decreased by greater than 95% leading to termination of the test. Animals in both the treatment and control tanks were fed unspiked diet during the depuration phase. At days 1, 4, 7, 14 and 21 of the depuration phase five fish from each of the tanks were sacrificed for analysis of the tissue concentrations of the different forms of cardanol. Animals in both tanks were fed at a rate of 2% body weight once per day. No mortalities occurred in the tanks during the study and no unusual behavior was noted. The growth of the fish in the control tanks during the study was consistent with that normally observed for the test species (see Table 4).


 


The measured concentrations of the different forms of cardanol in the fish tissues during the uptake and depuration phases allowed a series of endpoints to be calculated as shown in Table 5, namely:


 



  • Cfood (chemical concentration in the food; μg/g)

  • α (substance assimilation efficiency)

  • k2 (overall depuration rate constant; day-1)

  • k2g (growth-corrected depuration rate constant; day-1)

  • C0,m (measured time zero concentration, the concentration in fish at the end of uptake; μg/g)

  • C0,d (derived time zero concentration of depuration phase; μg/g)

  • t1/2g (growth-corrected half-life; day)

  • BMFK (kinetic dietary BMF)

  • BMFKg (growth-corrected kinetic dietary BMF)

  • BMFKgL (lipid-corrected growth-corrected kinetic dietary BMF)


 


During the uptake phase levels of the three forms of cardanol increased in the animals receiving the spiked diet, the mean concentrations on day 14 (the measured time zero concentrations) being 7.46 µg/g for the monoene form, 3.91 µg/g for the diene form and 2.80 µg/g for the triene form (see Table 5). The test substance constituents were not detectable in fish from the control tank which received unspiked diet.


 


During the depuration phase elimination of the accumulated forms of cardanol was rapid and 95% loss had been measured for both the diene and triene forms by day 7 and by day 14 for the monone form (where elimination after 7 days was 84%). All forms of cardanol were below the limit of quantification (<0.10 µg/g) by either depuration day 14 (diene and triene forms) or day 21 (monoene form). The time to reach 50% clearance of the monoene, diene and triene forms of cardanol was 1.653, 0.929 and 0.676 days respectively (see Table 5). The resulting derived time zero concentrations for cardanol monoene, diene and triene were 8.28 µg/g, 4.64 µg/g and 3.30 µg/g respectively.


 


The BMFK (growth corrected kinetic dietary biomagnification factors) values were 0.00946, 0.00445 and 0.00318 for the monoene, diene and triene forms of cardanol. The BMFKgL (lipid corrected growth corrected kinetic dietary biomagnification factors) values were 0.0387, 0.0182 and 0.0130 for the monoene, diene and triene forms of cardanol. The lipid content of the fish determined on Days 14 and 21 of the depuration phase had not changed from that measured at the start of test.


 


To assess the extent to which the different forms of cardanol present in Distillation Residue Grade exhibit bioaccumulation in fish two approaches have been used, namely:


 



  • Calculating tentative BCFs using a model predicted uptake rate constant (k1) and the depuration rate constant (k2) determined from the dietary exposure study.


 



  • Using the depuration rate constant (k2) as a surrogate for a BCF value based on the approach described in Brooke and Crookes (2012), in which the use of k2 as an alternative metric for assessment of the B (BCF >2,000 L/kg) or vB (BCF >5,000 L/kg) properties of a substance.


 


The tentative BCF values (L/kg) derived from the studies were 837-893, 414-478 and 280-362 for the monoene, diene and triene forms of cardanol respectively (based on the methods of Brooke and Crookes, 2012 and Inoue et al 2012) which are significantly below the 2000 L/Kg threshold for a substance to be considered to be bioaccumulative.


 


The overall depuration rate (k2) constants derived in the study were 0.428, 0.755 and 1.034 for the monoene, diene and triene forms of cardanol respectively. All these values were higher than the critical < 0.178 (no lipid normalisation) value for k2 which corresponds to the threshold BCF value (no lipid normalisation) of >2000 (Brookes and Crookes, 2012). Using the regression equation between log BCF and log K2 given in Table 3 (for data with no lipid normalisation) the estimated BCF values (L/kg) for the different forms of cardanol are 672, 333 and 245 for the monoene, diene and triene forms of cardanol respectively. These results indicate that the different forms of cardanol (monoene, diene and triene) did not markedly bioaccumulate and the estimated BCFs 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.


 


On the basis of the data generated in the study for the different forms of cardanol the test substance Distillation Residue Grade is not considered to be bioaccumulative.

Description of key information

A definitive dietary study in which rainbow trout were fed spiked diet at 1000 mg/kg has been carried out according to the OECD TG305 method. An uptake phase of 14 days was followed by a depuration phase of 21 days during which elimination of the accumulated forms of cardanol was rapid.


 


The data from the study resulted in BMFK (growth corrected kinetic dietary biomagnification factors) values of 0.00946, 0.00445 and 0.00318 for the monoene, diene and triene forms of cardanol. The BMFKgL (lipid corrected growth corrected kinetic dietary biomagnification factors) values were 0.0387, 0.0182 and 0.0130 for the monoene, diene and triene forms of cardanol.


 


Bioconcentration Factors (BCFs) were also derived using the data generated in the study based on the methods of Brooke and Crookes, 2012 and Inoue et al, 2012). The tentative BCF values (L/kg) derived from the study were 837-893, 414-478 and 280-362 for the monoene, diene and triene forms of cardanol respectively which are significantly below the 2000 L/Kg threshold for a substance to be considered to be bioaccumulative.


 


On the basis of the data generated in the study the different forms of cardanol and the test substance Distillation Residue Grade are not considered to be bioaccumulative.

Key value for chemical safety assessment

BCF (aquatic species):
900 L/kg ww
BMF in fish (dimensionless):
0.039

Additional information

A reliable without restrictions (Klimisch Code 1) definitive dietary study has been carried out according to the OECD TG305 method using a total of 50 juvenile rainbow trout in each of the control and treatment tanks. It was planned to use the aqueous exposure route in the study for ease of comparison of the resulting data with that for Distilled Grade, but attempts at two different test laboratories were not successful due to issues with the analysis of the test substance and maintenance of stable, measurable water concentrations. The dietary exposure study incorporated a 14 day uptake phase where test animals were exposed to diet spiked with 1000 mg/kg Distillation Residue Grade followed by a 21 day depuration phase where animals were fed unspiked diet. Fish in the control tank were fed unspiked diet throughout the study. Fish were sampled on day 14 of the uptake phase and on days 1, 4, 7, 14 and 21 of the depuration phase. The concentrations of the three key forms of cardanol present in the test substance (monoene, diene and triene) were measured in the spiked diet and fish tissues.


During the uptake phase levels of the three forms of cardanol increased in the animals receiving the spiked diet. The mean concentrations on day 14 (the measured time zero concentrations) were 7.46 µg/g for the monone form, 3.91 µg/g for the diene form and 2.80 µg/g for the triene form. These constituents of the test substances were not detectable in fish from the control tank which received unspiked diet. During the depuration phase elimination of the accumulated forms of cardanol was rapid and 95% loss had been measured for both the diene and triene forms by day 7 and by day 14 for the monone form (where elimination after 7 days was 84%). All forms of cardanol were below the limit of quantification (<0.10 µg/g) by either depuration day 14 (diene and triene forms) or day 21 (monoene form). The time to reach 50% clearance of the monoene, diene and triene forms of cardanol was 1.653, 0.929 and 0.676 days respectively.