2-Propenoic acid, methyl ester, reaction products with mixed O,O-bis(branched and linear pentyl and iso-Bu) phosphorodithioates

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:

2012-06-13 to 2012-09-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Bioconcentration test of chemical substances in fish and shellfish (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kampokihatsu N.110331009, March 31, 2011).

Deviations:

no

GLP compliance:

yes

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms: for fish from each concentration level sampled on days 0, 4, 7, 14, 21 and 28 of the exposure period. Remaining fish stored in a freezer for 3 months and then discarded unless additional analysis required. Daily observations on appearence, swimming and feeding behaviour.Lipid content measured in up to 5 fish from each test concentration and the control at start and end of exposure period- Sampling intervals/frequency for test medium samples: Test water from each concentration level sampled on days 0, 4, 7, 14, 21 and 28 of the exposure period. Dissolved oxygen and temperature measured on test water sampling days, pH measured at start and end of exposure period- Sample storage conditions before analysis:- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):Test water: 100 mL (high concentration level) or 1000 mL (low concentration level) was passed through an Empore disk C18*47 mm (preconditioned with approximately 10 mL acetonitrile and approximately 10 mL ultra pure water) under vacuum. 2 x 4.9 mL acetonitrile was added to the filtrate and the aqueous phase discarded each time. Acetonitrile was added to make up the remaining elutant to 10 mL volume and the sample was analysed by LC/MS.Test substance in fish:Two of the exposed fish were diced and homogenised in 4 bursts of 1 minute each at 8000 rpm.5 g of the sample was placed in 20 mL of acetonitrile and homogenised for 3 minutes at 8000 rpm.The remaining sample was frozen.The sample was filtered and divided into the acetonitrile layer and residue. The residue was put back through the acetonitrile extraction step and the second acetontrile layer added to the original extract. The remaining residue was discarded.The acetontrile extraction was made up to 50 mL in total and a 10 mL sample was taken and diluted with 10 mL of ultra pure water.The sample was filtered through an Inertsep C18-B*2 (preconditioned with 5 mL acetonitrile and 5 mL ultra pure water).The eluent (approximately 4.8 mL acetontrile) was made up to 5 mL, passed through Inertsep NH2*3 (preconditioned with 5 mL acetonitrile) and the sample was analysed by LC/MS.

Vehicle:

yes

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

Tetrahydrofuran: 40 ppm (v/v)The test material was dosed via the feed solution.High concentration level: 0.5 g of the test substance was dissolved in up to 1000 mL tetrahydrofuran to produce a 500 mg/L test concentrationLow concentration level: 100 mL of the high concentration feed solution was dissolved in up to 1000 mL tetrahydrofuran to produce a 50 mg/L test concentration

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM- Common name: Carp- Source: Niikura Fish Farm, 1217, Shimoya, Isehara, Kanagawa 259-1123, Japan- Age at study initiation (mean and range, SD): approx 1 year- Length at study initiation (lenght definition, mean, range and SD): 8 ± 4 cm- Weight at study initiation (mean and range, SD): ca. 5 g- Health status: visual inspection of health- Description of housing/holding area: Flow-through aquarium. 1000 L/day, (turnover rate: 20 volumes/day)- Feeding during test- Food type: Babygold, Kyorin- Amount: equivalent to 1.5% of body weight. Feed solution at 40 mL per day- Frequency: dailyACCLIMATION- Acclimation period: 2012-05-25 to 2012-06-28- Acclimation conditions (same as test or not): same- Type and amount of food: equivalent to 1.5% of body weight- Feeding frequency: daily - Health during acclimation (any mortality observed): < 5% mortality, no medication applied.

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

28 d

Total depuration duration:

0 d

Hardness:

53 mg CaCO3/L

Test temperature:

24± 2°C

pH:

6.0 - 8.5

Dissolved oxygen:

>= 60% of satuartion (>= 5 mg/L at 24°C)

TOC:

0.4 mg/L

Salinity:

Not applicable

Details on test conditions:

TEST SYSTEM- Vessel: 56 L glass aquarium, 50 L water- Aeration: Continuous- No. of organisms per vessel: 32 /50 L water for test concentrations and 12/50L water for control- No. of vessels per concentration (replicates): 1- No. of vessels per control / vehicle control (replicates): 1TEST MEDIUM / WATER PARAMETERS- Source/preparation of dilution water: Dechlorinated water produced by treating tap water with activated charcoal filter and sodium thiosulfate.- Particulate matter: below detection limit- Metals: below detection limit- Pesticides: below detection limit- Chlorine: below detection limit- Alkalinity: 41 mg CaCO3/L- Conductance: 15 mS/m- Holding medium different from test medium: No- Intervals of water quality measurement: 6 monthsOTHER TEST CONDITIONS- Adjustment of pH: No- Photoperiod: 16 hours light - Light intensity: 400 - 700 nmAcute toxicity test with Medaka (Oryzias lapites)- Test concentrations: > 2 mg/L- Results used to determine the conditions for the definitive study: yes (1/100 and 1/1000 of 96h LC50)

Nominal and measured concentrations:

Nominal: 0.02 and 0.002 mg/L

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS FOR CALCULATION OF BCFBCF calculated using equation:BCFn = C f,n / C w,nSteady-state BCFss is calculated using the equation:BCF ss = Cf,s / Cw,s WhereBCF n is the BCDF after n daysCf,n is the concentration in fish after n days (µg/g)Cw,n is the mean concentration of the test substance in water over n days (mg/L)Cf,s is the mean concentration in fish at steady state (µg/g)Cw,s is the mean concentration in test water at steady state (mg/L)Steady-state is considered to be reached when 3 consecutive measurements of BCF at intervals of 48 hours or longer are within 20% of each other. Cf,s is calculated from the from the last 3 consecutive values and Cw,s is the average of all values during the steady-state period.When the BCF values are all less than 100 it is considered the steady-state is reached.

Lipid content:

1.8 %

Time point:

start of exposure

Remarks on result:

other: n=3, 1.5% - 2.3%

Lipid content:

3.4 %

Time point:

end of exposure

Remarks on result:

other: n=3, 1.6% - 5.2%

Lipid content:

2.3 %

Time point:

start of exposure

Remarks on result:

other: n=5, 1.5% - 3.7%

Lipid content:

2.8 %

Time point:

end of exposure

Remarks on result:

other: n=5, 1.6% - 5.2%

Type:

BCF

Value:

6 - 14 dimensionless

Basis:

whole body w.w.

Time of plateau:

14 d

Calculation basis:

kinetic

Remarks on result:

other: Component B, High dose level

Remarks:

Conc.in environment / dose:0.02 mg/L

Type:

BCF

Value:

10 dimensionless

Basis:

whole body w.w.

Time of plateau:

14 d

Calculation basis:

steady state

Remarks on result:

other: Component B, High dose level

Remarks:

Conc.in environment / dose:0.02 mg/L

Type:

BCF

Value:

9 - 21 dimensionless

Basis:

whole body w.w.

Time of plateau:

14 d

Calculation basis:

kinetic

Remarks on result:

other: Component C, High dose level

Remarks:

Conc.in environment / dose:0.02 mg/L

Type:

BCF

Value:

7 - 15 dimensionless

Time of plateau:

14 d

Calculation basis:

kinetic

Remarks on result:

other: Component D, High dose level

Remarks:

Conc.in environment / dose:0.02 mg/L

Type:

BCF

Value:

8 - 26 dimensionless

Basis:

whole body w.w.

Time of plateau:

14 d

Calculation basis:

kinetic

Remarks on result:

other: Component F, High dose level

Remarks:

Conc.in environment / dose:0.02 mg/L

Details on kinetic parameters:

Not measured

Metabolites:

Not monitored. The substance is a UVCB.

Results with reference substance (positive control):

Not applicable

Details on results:

Validity of the testThere were no deviations or specific factors that might have affected the reliability of the results. The results of the study fulfill all the validity criteria.Monitoring of test conditionsConditions of the test fish were as follows: - The dissolved oxygen concentrations in all aquaria were maintained at >=60% saturation level of 7.3-8.3 mg/L, and the water temperature within 24±2°C range of 24.1 -24.4°C, bioth fulfilling the criteria of the test conditions. - The pH values weer 7.0 - 7.1, being in the proper range for fish rearing (6.0 - 8.5) - Surviving fish showed no abnormality in appearence, swimming or feeding behaviour. Therefore, the test fish were confirmed to be reared appropriately. No occurrence of mortality or abnormality was observed. - The difference between the average lipid content values (n=3) at the start and the end of the exposure period were not within the range of ±25%. So lipid contents of another two fish were measured and the average lipid content values (n=5) at the start of the exposure period were compared with those at the end of it. As a result the mean lipid content value measured at the end of the exposure period fell within ±25% of the mean value at the start. Concentration of test substance in test waterThe mean concentrations of the test substance in water can be found in Table 1.The variation of the test substance concentrations in the test water were within ±20% of the measured mean for components B, C and D at both concentration levels.As for the component F at both concentration levels, the variation of the concentration in the test water was not maintained within ±20% of the measured mean.However, it was within ±20% of the measured mean from day 14 to 28 of the exposure, in which verification of the steady state attainment was required.BCF of test substance1) Analysed componentsThe BCF and steady-state BCF values are shown in Table 2.The variation of the bioconcentration factor (average) of the high concentration level for component B at the three consecutive measurements with at least 48 hours interval fell within ±20%, and the str=eady state was confirmed during day 28 of the exposure period.The variations of the mean BCF from three consecutive determinations separated by intervals of more than 48 hours could not be confirmed to fall within ±20% at the low concentration level for component B and at both concentration levels for components C, D and F. However, the BCF were considered to have reached the steady-state since all of the values were shown to be less than 100 during the exposure period.2) Monitored componentsFor monitored components A, E and G, bioconcentration factors were estimated from the analytical chromatograms of fish sampled on day 28.A peak was observed at the high concentration level for component G , so the BCF (average) was calculated to be 21. The nominal concentration was used to calculate the BCF so correction by recovery rate was not performed.No peak attributable to the monitored components were observed inthe chromatograms of the test fish at the low concentration level for component G, and at both concentration levels for components A and E.It can be concluded from the results that the bioconcentration potential of the test substance in fish is low.

Table 1: Mean concentration of test substance in water

Component	Mean concentration in water (mg/L)
	High concentration level	Low concentration level
B	0.0163	0.00165
C	0.0161	0.00169
D	0.0178	0.00182
F	0.0197	0.00205

Table 2 BCF and BCFss

Component	High concentration level		Low concentration level
	BCF	BCFss	BCF	BCFss
B	6 – 14	10	<62-<66	<66
C	9 – 21	≤21	<61-<65	<65
D	7 – 15	≤15	<58-<62	<62
F	8 - 26	≤26	<46-<52	<52

Additional results tables can be found in the attached document.

Validity criteria fulfilled:

yes

Conclusions:

The BCF values for all quantifiable components, and for non-quantifiable components where a peak was observed in the LC/MS chromatograms, are all less than 100. It can be considered that the steady state was reached during the course of the 28-day exposure period. It can be concluded from the results that the bioconcentration potential of the test substance in fish is low.

Executive summary:

Test Guidance

The study was performed in accordance with the test guideline; Bioconcentration test of chemical substances in fish and shellfish (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kampokihatsu N.110331009, March 31, 2011).

Method

Carp (Cyprinus carpio) were exposed in a continuous flow-through system to the test material at doses of 0.2 and 0.02 mg/L (32 fish/dose level) for 28 days via feed. Dose levels were determined from an acute toxicity test with Oryzias latipes (EC50 > 2 mg/L). A control group of 12 fish was run under the same conditions, minus the addition of the test material to feed, to the test concentrations. Test substance concentrations in both water and fish were measured on days 0, 4, 7, 14, 21 and 28 of the exposure period, with daily observations on appearence, swimming and feeding behaviour. Lipid content was measured in up to 5 fish from each test concentration and the control at start and end of exposure period.

Test substance concentrations were measured using LC/MS and the molecular ion reference for four quantifiable and three non-quantifiable components of the test substance:

Quantifiable ion - Component B: 329.0 m/z Component C: 343.1 m/z Component D: 357.1 m/z Component F: 385.1 m/z

Monitor ion - Component A: 315.0 m/z Component E: 371.1 m/z Component G: 399.1 m/z

The concentrations of each component are expressed in terms of the weight concentrations of the substance without regard to the relative proportion of the component in the composition of the test substance.

The BCF and steady-state BCF (BCFss) were determined by comparison of the concentration of the test substance in the fish against the concentration of test substance in the water.

Results

The lipid content of carp was measured as 2.3% (n=5, 1.5 - 3.7%) at the start of the exposure period and 2.8% (n=5, 1.6 - 5.2%) at the end of the exposure period. The bioconcentration factors are:

Component	High concentration level		Low concentration level
	BCF	BCFss	BCF	BCFss
B	6 – 14	10	<62-<66	<66
C	9 – 21	≤21	<61-<65	<65
D	7 – 15	≤15	<58-<62	<62
F	8 - 26	≤26	<46-<52	<52

For monitored components A, E and G, bioconcentration factors were estimated from the analytical chromatograms of fish sampled on day 28. A peak was observed at the high concentration level for component G , so the BCF (average) was calculated to be 21. The nominal concentration was used to calculate the BCF so correction by recovery rate was not performed. No peak attributable to the monitored components were observed inthe chromatograms of the test fish at the low concentration level for component G, and at both concentration levels for components A and E. Conclusion It can be concluded from the results that the bioconcentration potential of the test substance in fish is low.

Description of key information

The BCF values for all quantifiable components, and for non-quantifiable components where a peak was observed in the LC/MS chromatograms, are all less than 100. It can be considered that the steady state was reached during the course of the 28-day exposure period. It can be concluded from the results that the bioconcentration potential of the test substance in fish is low.

Key value for chemical safety assessment

BCF (aquatic species):

66 dimensionless

Additional information

A key study was performed in accordance with the test guideline; Bioconcentration test of chemical substances in fish and shellfish (Japanese notification, Yakushokuhatsu 0331 No.7, Heisei 23.03.29 Seikyoku No.5, Kampokihatsu N.110331009, March 31, 2011).

Carp (Cyprinus carpio) were exposed in a continuous flow-through system to the test material at doses of 0.2 and 0.02 mg/L (32 fish/dose level) for 28 days via feed. Dose levels were determined from an acute toxicity test with Oryzias latipes (EC50 > 2 mg/L). A control group of 12 fish was run under the same conditions, minus the addition of the test material to feed, to the test concentrations. Test substance concentrations in both water and fish were measured on days 0, 4, 7, 14, 21 and 28 of the exposure period, with daily observations on appearence, swimming and feeding behaviour. Lipid content was measured in up to 5 fish from each test concentration and the control at start and end of exposure period.

Test substance concentrations were measured using LC/MS and the molecular ion reference for four quantifiable and three non-quantifiable components of the test substance:

Quantifiable ion - Component B: 329.0 m/z Component C: 343.1 m/z Component D: 357.1 m/z Component F: 385.1 m/z

Monitor ion - Component A: 315.0 m/z Component E: 371.1 m/z Component G: 399.1 m/z

The concentrations of each component are expressed in terms of the weight concentrations of the substance without regard to the relative proportion of the component in the composition of the test substance.

The BCF and steady-state BCF (BCFss) were determined by comparison of the concentration of the test substance in the fish against the concentration of test substance in the water.

The lipid content of carp was measured as 2.3% (n=5, 1.5 - 3.7%) at the start of the exposure period and 2.8% (n=5, 1.6 - 5.2%) at the end of the exposure period. The bioconcentration factors are:

Component	High concentration level		Low concentration level
	BCF	BCFss	BCF	BCFss
B	6 – 14	10	<62-<66	<66
C	9 – 21	≤21	<61-<65	<65
D	7 – 15	≤15	<58-<62	<62
F	8 - 26	≤26	<46-<52	<52

For monitored components A, E and G, bioconcentration factors were estimated from the analytical chromatograms of fish sampled on day 28. A peak was observed at the high concentration level for component G, so the BCF (average) was calculated to be 21. The nominal concentration was used to calculate the BCF so correction by recovery rate was not performed. No peak attributable to the monitored components were observed in the chromatograms of the test fish at the low concentration level for component G, and at both concentration levels for components A and E.

It can be concluded from the results that the bioconcentration potential of the test substance in fish is low.