Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

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:

15 July 1996 - 19 September 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 C (Bioaccumulation: Test for the Degree of Bioconcentration in Fish)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ‘Bioconcentration test of chemical substances in fish and shellfish’ specified in “The test method relating to new chemical substances” (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, July 13, 1974)

Deviations:

not specified

GLP compliance:

yes

Radiolabelling:

no

Details on sampling:

Frequency of analysis
-Analysis of the test substance in test water was performed twice weekly, 16 times in total during the exposure period for both concentrations tested (1 sample per analysis).
-Analysis of the test substance in test fish was performed 4 times in total at 2, 4, 6 and 8 weeks after the start of exposure for both concentrations tested (2 samples per analysis).
-Analysis of the test substance in control fish was performed before the start and at the end of exposure (2 samples per analysis).

PRE-TREATMENT OF ANALYTICAL SAMPLES
TEST WATER
Samples of test water, 50 mL from 0.1 mg/L concentration and 500 mL from 0.01 mg/L concentration, were collected from test vessels and pre-treated as follows to prepare samples for high performance liquid chromatography (HPLC) analysis:

Analytical Sample of Test Water
-450 mL of test water was sampled (only for 0.1 mg/L concentration).
-150 g of sodium chloride was added.
-2 mL of 20 mmol/L N, N-dimethylformamide containing lithium chloride was added.
-60 mL of chloroform was added and the sample shaken for 5 minutes.
-The aqueous phase was removed from the organic chloroform phase.
-The organic phase was subjected to dehydration filtration (using IPS filter paper).
-The sample was condensed to 1 - 2 mL (using a rotary evaporator at 50 °C with nitrogen purging).
-The sample was then made up to a total volume of 5 mL (20 mmol/L N, N-dimethylformamide containing lithium chloride, using volumetric flask).
-This pre-treatment provided a sample suitable for HPLC analysis.

TEST FISH
Test fish were sampled from the test vessels and pre-treated as follows to prepare samples for HPLC analysis:

Analytical Sample of Test Fish
-The body weight and length of the fish were measured before the fish were minced.
-80 mL of tetrahydrofuran was added and the sample homogenised (using Polytron for approximately 1 minute).
-The sample was washed with 40 mL of tetrahydrofuran and centrifuged at 7000 × gravity for 5 minutes.
-After centrifugation any residue was removed from the supernatant, which was then filtered using absorbent cotton.
-The sample was made up to 300 mL in volume with tetrahydrofuran (using volumetric flask).
-A 50 mL aliquot was removed and 1 mL of 20 mmol/L N, N-dimethylformamide containing lithium chloride was added.
-The sample was condensed to 1 - 2 mL (using rotary evaporator at 50°C with nitrogen purging).
-400 mL of deionized water was added, followed by 120 g of sodium chloride.
-40 mL of chloroform was added and the sample shaken for 10 minutes, followed by a further 40 mL addition of chloroform and a second 10 minute period of shaking.
-The aqueous phase was removed from the organic chloroform phase.
-The organic phase was subjected to dehydration filtration (using IPS filter paper).
-The sample was condensed to 1 - 2 mL (using a rotary evaporator at 50 °C with nitrogen purging).
-The sample was then made up to a total volume of 10 mL (20 mmol/L N, N-dimethylformamide containing lithium chloride, using volumetric flask).
-The sample was subjected to filtration using a 0.45 µm membrane filter.
-This pre-treatment provided a sample suitable for HPLC analysis.

Vehicle:

no

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

PREPARATION METHOD OF STOCK SOLUTION
Dispersant: HCO-40
Method of preparation: The test substance and HCO-40 (the volume was 50-fold that of the test substance) were dissolved in tetrahydrofuran. After distillation of tetrahydrofuran, they were dissolved into deionized water to prepare 1000 mg/L stock solution.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp.
- Source: Sugishima Fishery, 123-2, Gunchikuichibancho, Yatsushiro-shi, Kumamoto 866, Japan.
- Length at study initiation: Mean 9.3 cm.
- Weight at study initiation: Mean 20.4 g.
- Health status: Healthy - any abnormal fish were removed during the quarantine period.
- Description of housing/holding area: Kept in a cultivation vessel under flow-through conditions.
- Feeding during test: Yes. Feeding was stopped on the day before sampling.
- Food type: Mixed feed pellets for carp (Nippon Formula Feed Manufacturing Co., Ltd.).
- Amount: A total amount of approximately 2 % of their body weight per day.
- Frequency: Twice daily.

ACCLIMATION
- Acclimation period: 64 days.
- Acclimation conditions: After a 3 day quarantine period, fish were transferred into acclimatisation vessels following medicated bath treatment for parasitic extermination. After re-treatment in a medicated bath, the fish were then acclimated and those showing signs of abnormality were removed during this period. The fish were held for 37 days under flow-through conditions at the water temperature of 25 ± 2 °C. They were then transferred to the test vessels and held for further 27 days after medicated bath treatment at the same temperature under flow-through conditions.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

8 wk

Hardness:

No data

Test temperature:

25 ± 2 °C

pH:

No data

Dissolved oxygen:

7.0 - 7.9 mg/L

TOC:

No data

Salinity:

n/a

Details on test conditions:

BIOCONCENTRATION TEST
Testing and environmental conditions
- Supply method of test water: Flow-through apparatus assembled at the laboratory was used.
- Test vessels: 100 L glass aquaria.
- Volume of test water: Stock solution and test water were mixed at the ratio of 2 mL/minute, and 800 mL/minute, respectively; a total volume of 1155 L/day was supplied to the test vessels.
- Tank for stock solution: 25 L glass bottle.
- Number of test fish: 15 fish (at the start of the exposure). 5 fish were used at the start of the exposure in the control vessel.

Test water
- Type: Ground water pumped up into the test laboratory.
- Water quality: Analysis of the groundwater showed all components to be present at less than the levels specified in the guideline.

Preparation method of stock solution
-Dispersant: HCO-40
-Method of preparation: The test substance and HCO-40 (the volume was 50-fold that of the test substance) were dissolved in tetrahydrofuran. After distillation of tetrahydrofuran, they were dissolved into deionized water to prepare 1000 mg/L stock solution.

Concentration 0.1 mg/L: The test substance and HCO-40 (the volume was 50-fold that of the test substance) were dissolved in tetrahydrofuran. After distillation of tetrahydrofuran, they were dissolved into deionized water to prepare stock solution of 40 mg/L of test substance concentration in stock solution tank.
Concentration 0.01 mg/L: The test substance and HCO-40 (the volume was 50-fold that of the test substance) were dissolved in tetrahydrofuran. After distillation of tetrahydrofuran, they were dissolved into deionized water to prepare stock solution of 4 mg/L of test substance concentration in stock solution tank.
Control solution: HCO-40 was dissolved in deionized water to prepare stock solution of 2000 mg/L of HCO-40 concentration in the stock solution tank.

Test concentrations
The test substance concentration took into account the preliminary 48-hour LC50 and analytical sensitivity of the test substance.

Observations and measurements
- Observation of test fish: Health condition, etc., of the test fish were visually observed twice a day.
- Volume of test water: Measured and recorded once daily using a measuring cylinder.
- Test temperature: Measured and recorded once daily using an alcohol thermometer.
- Dissolved oxygen concentration: Measured and recorded twice weekly using dissolved oxygen analysers.
- Others: Excrement and smears on the aquaria wall, etc., were removed approximately once daily during the test period.

PRELIMINARY ACUTE TOXICITY TEST
Test method
The test was conducted in accordance with the method described in "Testing methods for industrial waste water: Acute toxicity test in fish" (section 71 of JIS K 0102-1993).

Test fish
-Species: Japanese killifish (Oryzias latipes).
-Source: Nakajima Fishery, Daimyoujin, Nagasumachi, Tamana-gun, Kumamoto 869-01, Japan.
-Quarantine conditions: Abnormal fish were removed after visual inspection upon their arrival. Remaining fish were treated with medication in a cultivation vessel and then held for 31 days under flow-through conditions.
- Acclimatisation conditions: After cultivation, fish were transferred into acclimatisation vessels and treated with a medicated bath. Fish showing signs of abnormality were removed during this period. The fish were held for 13 days under flow-through conditions at the water temperature of 25 ± 2 °C. Subsequently, selection and treatment in a medicated bath were performed again; the fish were then held for 32 days under flow-through conditions.
- Body weight: mean 0.17 g.
- Body length: mean 2.8 cm.
-Screening: The fish used in the test came from the same lot, meeting the criteria in the mercuric chloride test specified by Kenji Tabata.

Test water
As above.

Test conditions
-Test vessels: Round glass aquaria.
- Volume of test water: 4 L/concentration.
-Water temperature: 25 ± 2 °C.
- Dissolved oxygen concentration: At the start of the exposure: 7.6 - 8.1 mg/L.
At the end of the exposure: 6.9 - 7.2 mg/L.
- pH: At the start of the exposure: 8.2.
At the end of the exposure: 7.8 - 8.0.
- Number of test fish: 10 fish/concentration.
- Exposure period: 48 hours.
- Exposure method: Semi-static (water renewal every 8 - 16 hours).

Preparation method of stock solution
As above.

Implementation of the test
-48-hour LC50 of the test substance ≥200 mg/L (calculated by Doudoroff method).

Nominal and measured concentrations:

Nominal concentrations of 0.1 and 0.01 mg/L.

Reference substance (positive control):

no

Details on estimation of bioconcentration:

The concentration of test material in the fish was calculated from the chromatograms generated by HPLC.

Lipid content:

3.8 %

Type:

BCF

Value:

7.9 - 41

Remarks on result:

other: Conc.in environment / dose:0.1 mg/L

Type:

BCF

Value:

25 - 164

Remarks on result:

other: Conc.in environment / dose:0.01 mg/L

Details on results:

Concentration
The mean test substance concentration in the test water was maintained at or above 87 % of the nominal concentration.

Observation of test fish
No abnormality was observed.

Concentration dependence of bioconcentration factors
The fish were observed to be slightly bluish in colour at the 0.1 mg/L concentration. It was considered that this was caused by a concentration-dependent tendency in the bioconcentration factor.

Table 1 Mean Concentration Values of the Test Substance in the Test Water

Nominal Concentration (mg/L)	Measured Concentration (mg/L)
	Week 2	Week 4	Week 6	Week 8
0.1	0.0946	0.0928	0.0904	0.897
0.01	0.00874	0.00898	0.00883	0.00871

 

Table 2 Bioconcentration Factors

Nominal Concentration (mg/L)	BCF
	Week 2	Week 4	Week 6	Week 8
0.1	7.9 13	41 24	2 41	27 18
0.01	25 40	99 67	103 164	117 75

 

Discussion

Proportions of each component in the test substance

The test substance is a multicomponent mixture with unknown structure; 3 peaks were detected in HPLC-GPC analysis. The molecular weights of these components were estimated to be approximately 1100 (peak 1), 100 - 200 (peak 2) and around 100 (peak 3). The proportions of each component were:

	Retention time (min)	Proportions of each component (%)
Peak 1	7.8	80.7
Peak 2	9.8	14.6
Peak 3	11.5	4.8

 

 

Bioconcentration factor of each component

The bioconcentration factor in this study was calculated for each component because a difference was noted in the bioaccumulation level of each component represented by one of the 3 peaks. Values from all components were used when calculating recovery rate for aquaria concentration, test water and test fish analysis.

 

Peak 1

Nominal Concentration (mg/L)	BCF
	Week 2	Week 4	Week 6	Week 8
0.1	5.7 6.8	13 9.9	9.1 10	10 8.6
0.01	43 44	60 57	56 72	67 53

 

Peak 2

Nominal Concentration (mg/L)	BCF
	Week 2	Week 4	Week 6	Week 8
0.1	9.6 20	49 30	27 48	34 22
0.01	63 74	128 130	167 170	151 79

 

Peak 3

Nominal Concentration (mg/L)	BCF
	Week 2	Week 4	Week 6	Week 8
0.1	118 192	495 252	200 504	327 184
0.01	516 685	1140 595	1050 1850	1380 1080

 

Half-life period was found to be approximately 4 days based on the results from an excretion test which was performed focusing on peak 3 at the 0.01 mg/L concentration.

Identification was attempted by gas chromatography-mass spectrometry on this peak 3. However, analysis was not achieved.

Validity criteria fulfilled:

yes

Conclusions:

The bioaccumulation value at a concentration of 0.1 mg/L was 7.9 - 41.
The bioaccumulation value at a concentration of 0.01 mg/L was 25 - 164.

Executive summary:

A bioaccumulation study was carried out to evaluate the bioaccumulation potential of the test material. The study was conducted in accordance with the Japanese guideline “Bioconcentration test of chemical substances in fish and shellfish” specified in ‘The test method relating to new chemical substances’ (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, July 13, 1974), and OECD standardised guideline 305C.

Subsequent to an acute toxicity investigation in Oryzias latipes (48h LC50 200 mg) the BCF in Cyprinus carpio was determined over an 8 week exposure period. The exposure method was a continuous flow-through system.

 

The bioaccumulation value at a concentration of 0.1 mg/L was 7.9 - 41.

The bioaccumulation value at a concentration of 0.01 mg/L was 25 - 164.

Description of key information

A bioaccumulation study was carried out to evaluate the bioaccumulation potential of the test material. The study was conducted in accordance with the Japanese guideline“Bioconcentration test of chemical substances in fish and shellfish” specified in ‘The test method relating to new chemical substances’ (Kanpogyo No. 5, Yakuhatsu No. 615, 49 Kikyoku No. 392, July 13, 1974), and OECD standardised guideline 305C.The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Subsequent to an acute toxicity investigation in Oryzias latipes(48h LC50 200 mg) the BCF in Cyprinus carpio was determined over an 8 week exposure period. The exposure method was a continuous flow-through system.

 

The bioaccumulation value at a concentration of 0.1 mg/L was 7.9 - 41.

The bioaccumulation value at a concentration of 0.01 mg/L was 25 - 164.

Additionally, it was considered appropriate to run QSAR estimates for the identified components to determine the predicted bioconcentration factor (BCF) values for each of the components individually. The compounds were entered into the BCFBAF v.3.01 Q(SAR) model within the EPI Suite™ software (v. 4.11 US EPA 2012) and screened for bioaccumulation potential.

The Log Kow and BCF values were calculated by EPI Suite™ for 17 of the 18 components in the substance. It was not possible to generate values based on the SMILES notation for one component, which accounts for ≥ 0 - ≤ 5 % w/w of the substance as a whole.

Based on the EPI Suite™ outputs a significant number of the components investigated were determined to potentially fulfil the criteria to be considered as bioaccumulative (B) or very bioaccumulative (vB). For several of these compounds the predicted BCF value was below the threshold and the only criterion fulfilled was therefore the log Kow screening criterion of >4.5; however, some of these are sufficiently large and the partition coefficient high enough that the compounds can be considered to have limited bioaccumulation potential.

Of the identified components for which EPI Suite™ predictions were made, only 12.34 % of the target substance was neither B nor vB. It can be seen that 21.7 % was potentially B/vB and 53.72 % predicted to be either B or B and vB. Since a significant number and proportion of the target substance components are predicted to be either B, or B and vB, nigrosine is considered to be potentially B and vB.

It is considered that a significant proportion of the substances identified in the analytical report may have the potential to bioaccumulate.

The report “1105411.UK0 – 1848: QSAR Estimation of the Bioaccumulation Potential of Components of Benzenamine, reaction products with aniline hydrochloride and nitrobenzene (Nigrosine)” is attached in section 13.2 of this dataset.