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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:
26 November 2015 - 16 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: "Method for Testing the Degree of Accumulation of Chemical Substances in Fish Body" stipulated in the "Testing Methods for New Chemical Substances"
Version / remarks:
March 31, 2011, No.0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29, 2011, No.5, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 110331009, Environmental Policy Bureau, Ministry of the Environment, Japan; partially revised, April 2, 2012, No. 0402-1, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare; March 28, 2012, No. 2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry, No. 120402001, Environmental Policy Bureau, Ministry of the Environment, Japan
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test
Deviations:
yes
Remarks:
To overcome the low solubility of the substance a vehicle was used. In view of the low concentrations in the main test this has not influenced the test results.
GLP compliance:
yes
Specific details on test material used for the study:
The substance is a mixture of two constituents: one is the Aldehyde type (which is the registered substance); the other one is the Acetal type (dimehoxy) with the name: 2-(3,3-dimethoxy-2,2dimethylpropyl)-6,6-dimethylbicyclo[3.1.1]hept-2-ene. In this test these constituent are present in 50.6 and 48.6%, respectively. The registered substance is the aldehyde and therefore the focus will be on the aldehyde constituent.
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms:
Analysis of test fish was performed five times during the test period. Four fish per treatment level were taken out at each sampling time and divided into two groups (two fish per group) because one fish was not enough for the analytical sensitivity of the test item. Test fish analyses were carried out at intervals of more than 48 hours, and the final exposure period was 28 days. Analysis of control fish was performed before the start of the experiment and after the experiment completion. Four fish were taken out at the sampling time and divided into two groups, and analyzed individually.

- Sampling intervals/frequency for test medium samples:
Test water was analyzed once before the first analysis of test fish and at the same time as analysis of test fish thereafter. One sample was analyzed at each sampling time.

- Lipid content in test fish
Lipid contents of fish were determined using control fish before and after the experiment. Six fish were taken out at each sampling time and divided into three groups (two fish per group). Lipid contents of each group were determined with gravimetric analysis after chloroform-methanol extraction.
Vehicle:
yes
Remarks:
N,N-dimethylformamide
Details on preparation of test solutions, spiked fish food or sediment:
Chemical name of vehicle (dispersant): N,N-dimethylformamide
Method: Preparation of stock solutions
Level 1(nominal concentration 10 µg/L): Test sample (500 mg) and HC0-40 (2.5 g, 5 times amount of the test sample) were dissolved in N,N-dimethylformamide to prepare 500 mg'L stock solution (1L).
Level 2 (nominal concentration 1 µg/L): The stock solution of Level 1 was dissolved in N,N-dimethylformamide to prepare 50.0 mg'L stock solution (IL).

Controls: HC0-40 (2.5 g) was dissolved in N,N-dimethylformamide to prepare 2500 mg'L stock solution for HC0-40 (IL).
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Common carp (Cyprinus carpio)
Reason for selection: The previous data conducted with this species can be compared and the size of this species is adequate for handling.
- Source: CERI Kunune
- Age at study initiation: Yearling fish
- Length at study initiation (length definition, mean, range and SD): 6.4-7.2 cm
- Weight at study initiation: control fish 6.09-9.46 g
- Weight at termination : control fish 6.84-9.94g
- Lipid content at test initiation: 3.97%
- Health status: No abnormality in behaviour or appearance were noted.
- Feeding during test: Feed for coloured carp ( protein content: 30%, Lipid content 4.0%). Manufactured by Kyorin Food corporation. Amount of feed corresponding to about 2% of total body weight was fed once or twice a day in halves. The fish were starved for 24 hours before sampling.

ACCLIMATION
The external disinfection was carried out in an aqueous solution containing OTC for fisheries (oxytetracycline hydrochloride, Kyoritsu Seiyaku) and sodium chloride (The Salt Industry Center of Japan). Thereafter fish were acclimatized in the following conditions.

- Acclimation period: Period: 52 days
- Acclimation conditions (same as test or not): yes
- Health during acclimation (any mortality observed): less than 5%.
Route of exposure:
aqueous
Justification for method:
aqueous exposure method used for following reason: the concentration of test substance is below the water solubility limit
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Remarks:
Groundwater from CERI premises
Total exposure / uptake duration:
28 d
Hardness:
14 mg//L ( total hardness Ca, Mg)
Test temperature:
Level 1(10 µg/L): 24.1-24.5°C
Level 2 (1 µg/L): 24.2-24.5°C
Control: 24.2-24.6°C
pH:
Level 1(10 µg/L): 7.6, 7.4
Level 2 (1 µg/L): 7.6, 7.6
Control: 7.7, 7.7
Dissolved oxygen:
Level 1(10 µg/L): 6.4-7.4 mg/L
Level 2 (1 µg/L): 6.5 – 7.4 mg/L
Control: 7.3 – 7.7 mg/L
TOC:
0.5 mg/L
Conductivity:
290 µS/cm
Details on test conditions:
TEST SYSTEM
- Test vessel:
Level 1 and 2- 70-L glass tank for volatile item
Control 70-L glass tank
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): 0.04 mL/min for stock solution and 2000 mL/min for dilution water, 2880 L/day of test water, were supplied.
- No. of organisms per vessel: 28 fish for test concentration and 14 in control.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Groundwater from the premises of CERI Kurume

Item Unit Measured value Determination limit
Total hardness (Ca, Mg) mg/L 14 1
Suspended solid mg/L < l 1
pH - 7.9 -
Total organic carbon mg/L 0.5 0.5
Chemical oxygen demand mg/L < l 1
Residual chlorine mg/L < 0.02 0.02
Ammonium ion mg/L < 0.1 0.1
Total cyanide mg/L < 0.05 0.05
Alkalinity mg/L 110 1
Electric conductivity µS/cm 290 1
Total mercury mg/L < 0.005 0.0005
Cadmium mg/L < 0.001 0.001
Chromium (VI) mg/L < 0.01 0.01
Lead mg/L < 0.001 0.001
Arsenic mg/L < 0.005 0.005
Iron mg/L 0.02 0.01
Copper mg/L < 0.001 0.001
Cobalt mg/L < 0.001 0.001
Manganese mg/L < 0.005 0.005
Aluminium mg/L < 0.02 0.02
Zinc mg/L < 0.1 0.1
Nickel mg/L < 0.001 0.001
Silver mg/L < 0.0001 0.0001
Organochlorine pesticides
1,2-Dichloropropane mg/L < 0.002 0.002
Chlorothalonil mg/L < 0.001 0.001
Propyzamide mg/L < 0.0008 0.0008
Chlomitrofen mg/L < 0.0001 0.0001
Simazine mg/L < 0.0003 0.0003

- Intervals of water quality measurement: Daily during the test period
Thiobencarb mg/L < 0.001 0.001
Organophosphorous pesticides
Diazinon mg/L < 0.0005 0.0005
Isoxathion mg/L < 0.0008 0.0008
Fenitrothion mg/L < 0.0003 0.0003
EPN mg/L < 0.0006 0.0006
Dichlorvos mg/L < 0.001 0.001
Iprobenfos mg/L < 0.0008 0.0008
PCB mg/L < 0.0005 0.0005
Coliform bacteria count - n.d. -
Fluorine mg/L 1.4 0.1
Anionic surfactant mg/L < 0.02 0.02

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14 hours light I 10 hours dark (artificial light of white fluorescent lamp)

RANGE-FINDING / PRELIMINARY STUDY

- justification for choice of test concentrations:
To determine the concentration of the test substance for the bioaccumulation test, a fish acute toxicity study was conducted in accordance with Japanese Industrial standards. In this test 10 Ricefish (Oryzias Latipes) per concentration, were exposed to nominal concentrations of 2.00 mg/L, 1.00 mg/L and 0.5 mg/L under semi static conditions for 96 hours. Species were selected for their similar sensitivity to common carp and test conditions were similar to that of the bioaccumulation test. The 96 hour LC50 values for PIBA was determined to be above test concentrations of 2.00 mg/L however taking into account of the toxicity of the dispersant (N,N-dimethylformamide), 2.00 mg/L was set as the upper limit of the test concentration.

- Results were used to determine the conditions for the definitive study

Nominal and measured concentrations:
Nominal concentrations: Level 1 (10 µg/L) and Level 2 (1 µg/L)
Measured concentration: :
Since the test item was a mixture of components A and B, two peaks were detected with GC-MS analysis. They were components A and B in elution order and were named Peaks 1 and 2, respectively.

Level 1 (10 µg/L) Peak 1: 9.25 (Standard deviation 0.483) µg/L
Peak 2: 10.4 (Standard deviation 0.30) µg/L
Level 2 (1 µg/L) Peak 1: 0.923 (Standard deviation 0.0446) µg/L
Peak 2: 0.992 (Standard deviation 0.0454) µg/L

Reference substance (positive control):
no
Details on estimation of bioconcentration:
Cw = {Cw(n-1)+ Cw(n)}/2 = average conc of test item in test water for 1st analysis of test fish
Cw ={Cw(n-2)+ Cw(n-1) + Cw(n)}/3 average conc of test item in test water for 2nd analysis of test fish
BCF = Cfish/Cw

Steady state:
Cws ={Cw(n-2)+ Cw(n-1) + Cw(n)}/3 for average conc of test item in test water
Cfs ={Cf(m-2)+ Cf(m-1) + Cf(m)}/3 for average conc of test item in test fish at a steady state
BCFss = Cfs/Cws
Lipid content:
3.97 %
Time point:
start of exposure
Lipid content:
4.82 %
Time point:
end of exposure
Key result
Conc. / dose:
1 µg/L
Temp.:
24.3 °C
pH:
7.9
Type:
BCF
Value:
< 2.7 dimensionless
Basis:
whole body w.w.
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: Aldehyde constituent (peak 1)
Key result
Conc. / dose:
10 µg/L
Temp.:
24.3 °C
pH:
7.9
Type:
BCF
Value:
< 27 dimensionless
Basis:
whole body w.w.
Time of plateau:
28 d
Calculation basis:
steady state
Remarks on result:
other: Aldehyde constituent (peak 1)
Conc. / dose:
1 µg/L
Temp.:
24.3 °C
pH:
7.9
Type:
BCF
Value:
411.48 dimensionless
Basis:
whole body w.w.
Time of plateau:
21 d
Calculation basis:
steady state
Remarks on result:
other: Acetal constituent (peak 2)
Remarks:
Average BCF over day 21, 24 and 28
Conc. / dose:
10 µg/L
Temp.:
24.3 °C
pH:
7.9
Type:
BCF
Value:
302.64 dimensionless
Basis:
whole body w.w.
Time of plateau:
21 d
Calculation basis:
steady state
Remarks on result:
other: Acetal constituent (peak 2)
Remarks:
Average BCF over day 21, 24 and 28
Metabolites:
A peak of converted product was detected on the chromatogram for fish analysis at Level 1 (10 µg/L), a,a,6,6-tetramethyl-bicyclo[3.1.l]hept-2-ene -2-propanol, which was Alcohol derivative of the Aldehyde constituent.This metabolite was only found in the fish samples but not in the test water and recovery test from test fish samples. BCF of this metabolite after 28 days was 32 (for which recovery rate could not be calculated because this product was not included in the pretreatment operation). The recovery was calculated using the test nominal concentration of the test item.
Details on results:
No abnormality in behavior or appearance was noted.

The change of lipid contents of after the experimental completion versus those before the experimental starting was +21% which was within ±25%.
Before initiation of experiment: 3.97%
After tennination of expe1iment: 4.82%

The validity criteria of test method

1) The temperature variation is less than ±2°C of set water temperature 25°C.
2) The concentration of dissolved oxygen does not fall below 60% of saturated concentration 8.1 mg/L at 25°C.
3) The concentration of the test item in the test tank is maintained within ±20% of the mean of the measured values during the test period.
4) The mortality or other adverse effects/disease inboth control and test group is not observed.

Concentration of the test item in test water (unit: µg/l)

Level

Peak

After 2 days

After 8 days

After 17 days

After 21 days

After 24 days

After 28 days

Average (standard deviation)

1

1

Aldehyde Constituent

8.76

9.64

9.04

8.70

9.82

9.56

9.25           

(0.483)

2

Acetal constituent

10.5

10.5

9.82

10.2

10.5

10.6

10.4

(0.30)

2

1

Aldehyde constituent

0.857

0.969

0.905

0.894

0.966

0.945

0.923 (0.0446)

2

Acetal constituent

0.926

0.999

0.947

1.03

1.02

1.04

0.992 (0.0454)

The measured concentrations of the test item in test water are shown above. Concentrations of the test item were maintained >= 86% of the nominal concentrations and the variations were within ±20% of the average measured concentrations.

Bioconcentration factors: (average value)

Level

Peak

After 8 days

After 17 days

After 21 days

After 24 days

After 28 days

1

1

Aldehyde constituent

<2.7

<2.7

<2.7

<2.7

<2.7

 

2

Acetal constituent

270

220

300

290

320

2

1

Aldehyde constituent

<27

<27

<27

<27

<27

 

2

Acetal constituent

250

320

420

410

411

Aldehyde constituent, Peak 1: In all test fish and in at least three successive analyses this peak was below the LOQ and therefore the BCF is < 27 l/kg and therefore the steady state was reached within 28 -days. The exact BCFss could not be calculated because the concentration in the fish was below the LoQ. This value will be used for the risk characterisation being the registered substance. In addition, a metabolite of this constituent was identified as alcohol derivative: α,α,6,6-tetramethyl-bicyclo[3.1.1 ]hept-2-ene-2-propanol. This metabolite had a low bioconcentration potential of 31-32 l/kg.

Acetal constituent, Peak 2: The BCFss was 400 l/kg and reached within 21 days because the variation of BCFs (average value) after 21, 24 and 28 days from the initiation of exposure were 0.2-7% within ±20%.

Validity criteria fulfilled:
yes
Remarks:
This is a guideline study. The substance is solubilised with a dispersant. In view of the low concentrations used 10 and 1 ug/l, this is not expected to have influenced the study results.
Conclusions:
Bioconcentration factor (BCFss) of Cyprinus carpio for Pinyl iso butyraldehyde as such was < 27 l/kg. Its main metabolite was the alcohol derivative: α,α,6,6-tetramethyl-bicyclo[3.1.1 ]hept-2-ene-2-propanol, with a low bioconcentration potential of 32. (The BCF of the acetal was set conservatively at 411 l/kg)
Executive summary:

An experimental study was performed to assess the bioconcentration of Pinyl Isobutyraldehyde in Cyprinus carpio under flow through conditions. The study was conducted in accordance with Japanese guidelines and GLP principles equivalent to OECD TG 305. A solvent was used due to the limited solubility of the substance. During the uptake phase, groups of 28 carp were exposed for 28 days to two concentrations of test substance dispersed with N-N-dimethylformamide and 14 fish exposed to untreated control. Nominal test concentrations of 1 and 10 µg/Lwere used and based on an acute toxicity test using Oryzias latipes (rice fish). Concentrations of the test item in water were maintained >= 86% of the nominal concentrations and the variations were within ±20% of the average measured concentrations.The lipid content of the fish (as mg/kg wet weight) at the end of the experiment did not differ from that at the start by more ± 25%. The BCF values for the Aldehyde constituent (the registered substance) was < 27 l/kg. Its metabolite being the alcohol derivative: α,α,6,6-tetramethyl-bicyclo[3.1.1 ]hept-2-ene-2-propanol, showed a BCF of 32. (The BCF values of Acetal constituent were roughly 300 and 400 at concentrations of 1 and 10 µg/L respectively and were reached within 21 days). Overall Pinyl isobutyraldehyde as the registered substance has no bioaccumulation potential (BCF < 100 l/kg).

Description of key information

An experimental study was performed to assess the bioconcentration of Pinyl Isobutyraldehyde (PIBA) in Cyprinus carpio under flow through conditions.The study was conducted in accordance with Japanese guidelines and GLP principles equivalent to OECD TG 305. A solvent was used due to the limited solubility of the substance. During the uptake phase, groups of 28 carp were exposed for 28 days to two concentrations of test substance dispersed with N-N-dimethylformamide and 14 fish exposed to untreated control. Nominal test concentrations of 1 and 10µg/L were used and based on an acute toxicity test using Oryzias latipes (rice fish). Concentrations of the test item in water were maintained >= 86% of the nominal concentrations and the variations were within ±20% of the average measured concentrations. The lipid content of the fish (as mg/kg wet weight) at the end of the experiment did not differ from that at the start by more ± 25%.The BCF values for the Aldehyde constituent (the registered substance) was < 27 l/kg. Its metabolite being the alcohol derivative:α,α,6,6-tetramethyl-bicyclo[3.1.1 ]hept-2-ene-2-propanol, showed a BCF of 32. (The BCF values of Acetal constituent were roughly 300 and 400 at concentrations of 1 and 10 µg/L respectively and were reached within 21 days). Overall Pinyl isobutyraldehyde as the registered substance has no bioaccumulation potential (BCF < 100 l/kg).

Key value for chemical safety assessment

BCF (aquatic species):
27 L/kg ww

Additional information

Value is '< ' , i.e. < 27 L/kg ww.