Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

Bioaccumulation: aquatic / sediment

Administrative data

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 E (Bioaccumulation: Flow-through Fish Test)

Deviations:

yes

Remarks:

fish were exposed to only one concentration of test substance

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

EC Number:

931-276-9

Cas Number:

114959-46-5

Molecular formula:

See information in Section 1.2.

IUPAC Name:

Benzoic acid, 2-hydroxy-, mono-C14-18-alkyl derivs., calcium salts (2:1)

Details on test material:

- Name of test material (as cited in study report): [CAS Number 114959-46-5]
- Physical state: Clear brown liquid
- Brixham test substance number: 03-0465
- Storage condition of test material: Ambient temperature

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Radiolabelling:

no

Sampling and analysis

Details on sampling:

No data

Test solutions

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

The dilution water was dechlorinated tap water, which had been passed through activated carbon, coarsely filtered to remove particulate matter and dechlorinated with sodium thiosulphate. Salts were added, as required, to maintain minimum water hardness levels and the treated water was then passed through an ultraviolet steriliser to a second set of 20 and 10 µm filters. The supply was then delivered to a temperature controlled header tank in the test laboratory set to the nominal test temperature of 15 ± 1 degrees Centigrade and was further filtered to 5 µm prior to delivery to the test vessels.

The pH and conductivity of the laboratory dechlorinated water supply was monitored every week day. Hardness, alkalinity and free and residual chlorine were measured once a week. Total ammonia was measured once a month.

The dechlorinated water supply was also monitored periodically for chemical oxygen demand, total organic carbon, total suspended solids, a range of cations and anions, trace metals, pesticides and other hydrocarbons. The sampling schedule for all of the dechlorinated water supply analyses are shown in Appendix 1 (attached).

Test organisms

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow ttrout
- Source: Houghton Springs Fish Farm, Winterbourne Houghton, Blandford Forum, Dorset DT11 0PD
- Length at study initiation: 41 to 78 mm (mean 58 mm, n=23)
- Weight at study initiation: 1.14 to 7.36 g (mean 3.31 g, n = 23)
- Feeding during test: Twice per day
- Food type: untreated commerical food, test material treated food or hexachlorobenzene treated food
- Amount: 3 % wet body weight per day based on bulk weight of 20 fish randomly selected from each tank

ACCLIMATION
- Acclimation period: holding period of at least 14 days in stock tanks; acclimation period of 7 days minimum at test temperature, acclimation period of 3 days minimum in test tanks
- Acclimation conditions (same as test or not): Acclimatised at test temperature
- Type and amount of food: Untreated commercial fish food at 3 % of wet body weight per day based on bulk weight of 20 fish from each tank (withheld for at least 24 hours prior to the commencement of the test)
- Medication: The fish were last medicated with 180 mg/L formalin more than 14 days prior to the test
- Health during acclimation (any mortality observed): There was less than 1 % mortality observed in the batch of fish in the 7 days prior to the start of the test

Study design

Route of exposure:

feed

Test type:

flow-through

Water / sediment media type:

not specified

Remarks:

freshwater

Total exposure / uptake duration:

10 d

Total depuration duration:

21 d

Test conditions

Hardness:

41.7 to 49.7 mg/L as calcium carbonate (see Table 11, attached)

Test temperature:

14.5 to 14.8 degrees Centigrade (see Table 14, attached)

pH:

7.32 to 7.58 (see Table 13, attached)

Dissolved oxygen:

9.2 to 10.0 mg/L (see Table 12, attached)

Corresponding air saturation values (quoted as percentage of the 100 % air saturation value of 10.06 mg/L at 15 degrees Centigrade) 91 to 100 %

TOC:

0.53 mg/L

Salinity:

0.5 to 1.5 o/oo (see Table 11, attached)

Details on test conditions:

POSITIVE CONTROL
The positive control, hexachlorobenzene, was supplied by Sigma-Aldrich Co Ltd, Fancy Road, Poole, Dorset BH12 4QH as a white powder of 99 % purity (batch number 80202022). The sample was stored at ambient temperature and used without adjustment for purity.

TEST CONDITIONS
A dynamic (flow through) test system was used for this study. The test apparatus was constructed of glass with a minimum of other materials (silicone tubing and sealant) in contact with the test solutions. The test vessels used throughout were all glass contruction, rectangular in shape and had a working volume of 105 L (915 mm long x 305 mm wide x 465 mm high).

The dilution water was fed from a header tank via flow control devices to 2 L glass aspirators and then to the test vessels. The nominal flow rate of the dilution water to each mixing chamber was 1000 mL/minute. Calibration of the dosing systemwas made by direct measurement of the flow rates into the 2 L glass aspirators twice per week throughout the study. The test solutions were gently aerated throughout the study.

TEST TEMPERATURE AND PHOTOPERIOD
The test was conducted at a nominal temperature of 15 ± 1 degrees Centigrade with a photoperiod of 16 hours light and 8 hours dark. There was a 20 minute dawn/dusk transition period.

The light intensity, measured by cosine receptor, was recorded at test vessel height, using a Skye Instruments unit SKL300 photometer with a matching sensor head type SKL310. Readings were taken at the centre and near the two ends of the test rig to determine the range of values along the entire length.

PHYSICAL PARAMETERS
Dissolved oxygen, pH and temperature measurements were made in all test vessels on day zero of the uptake phase. Dissolved oxygen and pH were then measured at least twice per week in all test vessels throughout the study. Temperature values were determined three times a week in all test vessels using a mercury-in-glass thermometer calibrated to 0.1 degrees Centigrade and conforming to BS593. A continuous record of the temperature was kept in both the test material and postive controls using an electronic recording system (platinium resistance sensor).

To ensure the correct dilution water was being supplied to the test rig, the salinity was measured once per week in the hexachlorobenzene control tank, throughout the study. In addition, the conductivity, alkalinity and total hardness of the hexachlorobenzene control tank were determined once per week throughout the study.

PREPARATION OF CONTROLS AND EXPOSED FEEDS
The study was carried out with a control feed, a test material treated feed (1000 mg/kg) and a positive control (hexachlorobenzene) treated feed (1000 mg/kg). Because the numbers of fish required for sampling in the control population of fish, two tanks were set up with approximately equal numbers of fish in each tank (see Appendix 2, attached). Those tanks are referred to as Control 1 and Control 2 for the purposes of reporting. The fish in the two control tanks all received the same control feed, which was prepared according to the same methods (and using the same quantity of solvent) as the treated feeds.

Food used in the study was a Finfish starter crumble number 2 (Ziegler Bros Inc, Pennsylvania, USA) of 0.841-1.19 mm. A single batch of each exposure diet was prepared prior to the start of the test.

PREPARATION OF TEST MATERIAL FOOD
A nominal 0.2 g of the test substance was measured into a 250 mL glass beaker and dissolved in 160 mL of hexane followed by stirring for approximately 2 minutes. The solution was then added to 200 g of pellet food in a glass 1 L storage container, and stirred for approximately 5 minutes using a glass rod. The nominal concentration of test substance in the food was 1000 mg/kg. The container was then left in an extraction fume hood for approximately 48 hours until the hexane had completely evaporated from the food. To ensure an even drying process, the food was stirred periodically. Once the drying process was complete, the food was refrigerated for the duration of the study.

PREPARATION OF THE HEXACHLOROBENZENE FOOD
A nominal 0.2 g of the hexachlorobenzene was measured into a 250 mL glass beaker and dissolved in 160 mL of hexane followed by stirring for approximately 2 minutes. The solution was then added to 200 g of pellet food in a glass 1 L storage container, and stirred for approximately 5 minutes using a glass rod. The nominal concentration of test substance in the food was 100 mg/kg. The container was then left in an extraction fume hood for approximately 48 hours until the hexane had completely evaporated from the food. To ensure an even drying process, the food was stirred periodically. Once the drying process was complete, the food was refrigerated for the duration of the study.

TEST PROCEDURE
The fish were randomly allocated to the test tanks and allowed to acclimate for a minimum of three days prior to the start of the study. The number of fish added to each tank is detailed in Appendix 2 (attached).

On day zero, 23 fish were obtained from the excess stock population for the baseline chemical analysis. These fish were terminated using a buffered 2 g/L tricaine methane sulphonate (MS222) solution and weighed and measured for standard length. The fish were then dissected and the complete digestive tract removed and discarded. The remainder of the viscera (such as the liver and spleen) was kept with the carcass for chemical analysis. Each fish was individually stored in a labelled glass vial and placed into a freezer at minus 80 degrees Centigrade until analysed.

The amount of food (3 % wet body weight per day) was calculated, 3 days prior to day zero, from groups of 20 fish randomly selected from each tank. The fish were fed twice per day throughout the duration of the study. The amount of food was adjusted after each sampling occasion to compensate for the numbers of fish sampled plus growth of the fish. All tanks were siphoned following the second feed to remove any uneaten food thus preventing the fish being exposed to the test substance via uptake from the water. The fish were monitored daily for any evidence of abnormal behaviour, appearance, or reduced feeding due to palatability issues.

At the end of the uptake phase, the fish were sampled and treated as detailed for day zero (see Appendix 2, attached). Subsequently all fish were fed untreated food following a recalculation of the feeding rate. The fish were periodically sampled and treated as detailed for day zero during the depuration phase until day 21.

Nominal and measured concentrations:

Test material treated feed: 1000 mg/kg
Hexachlorobenzene treated feed: 1000 mg/kg

Reference substance (positive control):

yes

Remarks:

Hexachlorobenzene

Details on estimation of bioconcentration:

The growth corrected depuration rate was calculated by subtraction of the growth rate from overall elimination rate:

k(depuration) = k(overall) - k(growth)

Chemical assimilation efficiency, dietary biomagnification factor (BMF) and growth-corrected half-life were calculated as shown in Appendix 5 (attached).

Results and discussion

Lipid content

Lipid content:

>= 4.12 - <= 10.71 %

Time point:

other: Day zero to Day 21

Remarks on result:

other: See Table 3 (attached)

Bioaccumulation factor

Bioaccumulation factor 1

Type:

other: concentration of test material in the fish

Value:

< 0.003 other: mg/kg

Basis:

whole body w.w.

Calculation basis:

other: see analytical method described in Appendix 3 (attached)

Remarks on result:

other: Mean value for Control 1 fish (C14, C16 and C18). Digestive tract removed and discarded, viscera kept with carcass for chemical analysis.

Remarks:

Conc.in environment / dose:zero mg/kg

Bioaccumulation factor 2

Type:

other: concentration of test material in fish

Value:

< 0.004 other: mg/kg

Basis:

whole body w.w.

Calculation basis:

other: see analytical method described in Appendix 3 (attached)

Remarks on result:

other: Mean value for exposed fish (C14). Digestive tract removed and discarded, viscera kept with carcass for chemical analysis.

Remarks:

Conc.in environment / dose:1000 mg/kg

Bioaccumulation factor 3

Type:

other: concentration of test material in fish

Value:

0.004 other: mg/kg

Basis:

whole body w.w.

Calculation basis:

other: see analytical method described in Appendix 3 (attached)

Remarks on result:

other: Mean value for exposed fish (C16). Digestive tract removed and discarded, viscera kept with carcass for chemical analysis.

Remarks:

Conc.in environment / dose:1000 mg/kg

Bioaccumulation factor 4

Type:

other: concentration of test material in fish

Value:

0.009 other: mg/kg

Basis:

whole body w.w.

Calculation basis:

other: see analytical method described in Appendix 3 (attached)

Remarks on result:

other: Mean value for exposed fish (C18). Digestive tract removed and discarded, viscera kept with carcass for chemical analysis.

Remarks:

Conc.in environment / dose:1000 mg/kg

Bioaccumulation factor 5

Type:

other: concentration of hexachlorobenzene in fish

Value:

< 0.02 other: mg/kg

Basis:

whole body w.w.

Calculation basis:

other: see analytical method described in Appendix 3 (attached)

Remarks on result:

other: Mean value for Control 2 fish. Digestive tract removed and discarded, viscera kept with carcass for chemical analysis.

Remarks:

Conc.in environment / dose:zero mg/kg

Bioaccumulation factor 6

Type:

other: concentration of hexachlorobenzene in fish

Value:

2.15 other: mg/kg

Basis:

whole body w.w.

Calculation basis:

other: see analytical method described in Appendix 3 (attached)

Remarks on result:

other: Mean value for exposed fish

Remarks:

Conc.in environment / dose:100 mg/kg

Bioaccumulation factor 7

Type:

BCF

Value:

1 470 dimensionless

Basis:

whole body w.w.

Calculation basis:

other: see Appendix 5 (attached)

Remarks on result:

other: Test material feed (C14)

Remarks:

Conc.in environment / dose:1000 mg/kg

Bioaccumulation factor 8

Type:

BCF

Value:

1 736 dimensionless

Basis:

whole body w.w.

Calculation basis:

other: see Appendix 5 (attached)

Remarks on result:

other: Test material feed (C16)

Remarks:

Conc.in environment / dose:1000 mg/kg

Bioaccumulation factor 9

Type:

BCF

Value:

2 427 dimensionless

Basis:

whole body w.w.

Calculation basis:

other: see Appendix 5 (attached)

Remarks on result:

other: Test material feed (C18)

Remarks:

Conc.in environment / dose:1000 mg/kg

Bioaccumulation factor 10

Type:

BCF

Value:

23 442 dimensionless

Basis:

whole body w.w.

Calculation basis:

other: see Appendix 5 (attached)

Remarks on result:

other: Hexachlorobenzene feed

Remarks:

Conc.in environment / dose:100 mg/kg

Depuration

Elimination:

not specified

Details on kinetic parameters:

See calculation methods described in Appendix 5 (attached)

Metabolites:

No data

Results with reference substance (positive control):

Triplicate samples of hexachlorobenzene treated food were analysed prior to the start and at the end of the study. The mean concentration of hexachlorobenzene in the treated food was 97 mg/kg. No hexachlorobenzene was detected in the control food throughout the study. Triplicate recovery samples of food fortified with hexachlorobenzene at the nominal 100 mg/kg were analysed on two occasions. The mean recovery from these samples was 102 % with a RSD of 5.6.

Details on results:

CONCENTRATION OF TEST MATERIAL IN TREATED FOOD
Triplicate samples of test material treated food were analysed prior to the start and at the end of the study. The mean concentrations of test material in the treated food were 77, 134 and 76 mg/kg for the C14, C16 and C18 homologues respectively. No test material was detected in the control food throughout the study. Triplicate recovery samples of food fortified with test material at the nominal 1000 mg/kg were analysed on two occasions. The mean recoveries from these samples were 109, 109 and 10 % with Residual Standard Deviation (RSD) of 5.6, 5.4 and 6.6 for the C14, C16 and C18 homologues respectively.

Reported statistics:

No data

Any other information on results incl. tables

CONCENTRATIONS OF TEST MATERIAL AND HEXACHLOROBENZENE IN THE FISH

The concentrations of test material and hexachlorobenzene are shown in Tables 1 and 2 (attached). The plots of ln(concentration) versus depuration day are shown in Figures 1 to 4 (attached). Summary bioaccumulation data are also attached.

IN LIFE FEEDING OBSERVATIONS

There were no observed adverse effects on feeding rates or visable symptoms of toxicity during the study.

DILUTION WATER AND FISH FOODS

Pesticide concentrations, trace metals, cations, anions and water quality parameters in routine samples of the dechlorinated water supplied the the laboratory are given in Tables 5, 6, 7 and 8 (attached). Analyses of the fish food diets are given in Tables 9 and 10 (attached). It is considered that none of the analytes were present in sufficient quantities to have affected the validity of the study.

LABORATORY WATER SUPPLY

During the study the pH of the laboratory water supply ranged from 7.51 to 7.86 and conductivity from 210 to 241 µS/cm. Alkalinity ranged from 21.8 to 23.2 mg/L and total hardness from 41.7 to 49.7 mg/L as calcium carbonate.

The free available residual chlorine and the total available residual chlorine were each determined on 4 separate occasions during the study. All determinations of the free available residual chlorine and the total available residual chlorine were less than 2 µg/L, which was the limit of detection (see Table 5, attached)

The ammonium-N, solids-suspended, Total Organic Carbon (TOC) and Chemical Oxygen Deman (COD) were each determined once during the test. The concentration of ammonium-N was 10.0 µg/L, suspended solids < 3.0 mg/L, TOC 0.53 mg/L and COD < 12 mg/L.

TEST SOLUTIONS

The data obtained on test solutions showed little variation during the whole test period. A summary of flow rates is shown in Table 15 (attached). In addition, the conductivity, alkalinity (as calcium carbonate) and total hardness (as calcium carbonate) were measured weekly throughout the study in one test vessel. The results are shown in Table 11 (attached).

LIGHT INTENSITY

The light intensity over the test rigs was recorded once during the study and three readings were taken over the test rig. The values, recorded as lux, ranged from 450 to 600.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Bioaccumulation factors were reported as 1470 (C14), 1736 (C16) and 2427 (C18) for fish exposed to feed containing test material. The corresponding bioaccumulation factor for fish exposed to feed containing hexachlorobenzene was 23442.