Registration Dossier

Administrative data

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 July 2008 - 18 August 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP. Tested as ammonium salt, detection system was specific for DONA.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)
Deviations:
yes
Remarks:
TOC measured in control was 2.1 mg/C/L at day 28 of the uptake phase but did not affect the fish and as such study integrity was not adversely affected by the deviation.
Qualifier:
according to
Guideline:
other: EC directive Annex IIID, Part C.13:1998
Deviations:
yes
Remarks:
TOC measured in control was 2.1 mg/C/L at day 28 of the uptake phase but did not affect the fish and as such study integrity was not adversely affected by the deviation.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MTDID 6675
- Substance type: single-constituent substance
- Physical state: clear colorless liquid
- Analytical purity: 29.9 ±0.0% in water
- Lot/batch no.: 140499-19/16
- Stability under test conditions: Stable in water at least 96 hours
- Storage condition of test material: At room temperature in the dark
Radiolabelling:
no

Sampling and analysis

Details on sampling:
Test Medium analysis
- Sampling intervals/frequency for test medium samples: Samples were taken after -1, 0, 1, 3, 7, 10, 13, 20, 30 and 34 days during uptake. 1 sample of control, low and high concentration solution was obtained at each sample interval.

BCF analysis
- Sampling intervals/frequency for test organisms: Samples were taken after 1, 3, 7, 10, 20, 30 and 34 day during uptake. At each sampling period 3 fish were taken from the control, 4 fish each from both the low and high concentration.

Lipid analysis
- Sampling intervals/frequency for test organisms: Samples were taken after 0 days for control and 30 days for test samples. 10 fish were taken on day 0 from the control and 10 fish each were taken on day 30 for low and high concentration.

- Sample storage conditions before analysis: Analysis of test media and fish used for BCF analysis were completed the same day as sampling, so storage was not applicable. Samples used for lipid extraction were stored in a deep-freezer until extraction.

- Details on sampling and analysis of test organisms and test media samples:
For the test media, test solutions were sampled from a central point in the test chamber using mechanical pipettes with disposable tips.

For the test organisms, fish were caught with a small net, quickly rinsed with untreated water, blotted dry and then instantly killed with a blow to the head, followed by a cervical incision. Fish used for BCF analysis were weighed and the tissue in each replicate was homogenized using a scalpel. Fish used for lipid extraction were weighed and pooled per concentration. Each lipid extraction fish was homogenized in dry-ice using a food processor.

Test media and fish for BCF analysis were measured by Ultra Performance Liquid Chromatography (UPLC). Samples for the lipid extraction were analysed using the Bligh and Dyer (BD) method (Protocol for this was not reported).

Test solutions

Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Method: ADONA consists of 29.9% active ingredient (a.i.) in water. Stock solutions of ADONA in ISO-medium were prepared twice a week. Stock concentrations were 330 and 3300 mg/L total product (100 and 1000 mg/L active ingredient). The stock solutions were dosed via a computer-controlled system (DaVinci) consisting of micro-dispensers (Gilson). Through this system the dosed volume from the supply stock entered a mixing flask separately from the medium supply. The medium was supplied via a flow meter and the flow rate was 16 L/h, allowing at least four volume replacements (100 L) through each aquarium each day. In the mixing flask the dosed stock volume and the medium were mixed under continuous stirring at a ratio of 1:1000. The whole system was checked daily.
- Controls: Test medium without test substance or other additives
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None, final test solutions were all clear and colorless.

Test organisms

Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Strain: Linneaus, 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Center, The Netherlands
- Age at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): 3.4 ± 0.3 cm
- Weight at study initiation (mean and range, SD): 1.08 ± 0.21 g
- Weight at termination (mean and range, SD): Not reported
- Feeding during test: Yes
- Food type: Pelleted fish food (Nutra 3.0 T, Trouw Nutrition B.V., Putten, The Netherlands)
Rough protein: 55%
Rough fat: 16%
- Amount: Ration was 2% of body weight per day. Recalculation of the feeding rate was performed after each sampling point.

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Acclimation conditions (same as test or not): same as test
- Feeding frequency: Daily with pelleted food fish (Nutra 3.0 T, Trouw Nutrition B.V., Putten, The Netherlands)
- Health during acclimation (any mortality observed): Mortality during seven days prior to the start of the test was less than 5%

Study design

Route of exposure:
aqueous
Test type:
flow-through
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
34 d

Test conditions

Hardness:
161 - 179 mg CaCO3/L
Test temperature:
21.7 - 23.2 °C
pH:
7.3 - 7.9
Dissolved oxygen:
7.1 - 9.1
TOC:
0.29 - 2.1
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 L (50x50x40 cm) tank consisting of stainless steel and covered by a removable Perspex plate
- Aeration: None
- Type of flow-through (e.g. peristaltic or proportional diluter): Peristaltic
- Renewal rate of test solution (frequency/flow rate): Four volume replacements per day, flow rate of 16 L/h
- No. of organisms per test concentration: 54
- No. of organisms per control: 43
- No. of vessels per concentration (replicates): Two
- No. of vessels per control (replicates): One
- Biomass loading rate: 0.15 g fish/L/day
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Conductance: 498 - 540 µs/cm
- Holding medium different from test medium: No
- Intervals of water quality measurement: pH, nitrate, nitrite and ammonia concentrations were measured once a week. Temperature was measured every day. In addition, pH and temperature were measured before transferring the fish to the test system. Total (dissolved organic carbon (TOC) in the control was measured once a week starting one day before introduction of the fish.
- Intervals of test medium replacement: Flow through with four 100 L replacements through each aquarium each day

OTHER TEST CONDITIONS
- Adjustment of pH: None
- Photoperiod: 16h light/ 8h dark

The experimental conditions of the study can be found in Table A.
Reference substance (positive control):
no

Results and discussion

Lipid content
Lipid content:
3 %
Time point:
other: 30 days
Remarks on result:
other: The control sample at the start of the exposure had 4% lipid.
Bioaccumulation factor
Type:
BCF
Value:
0.094 other: whole body w.w.
Time of plateau:
34 d
Calculation basis:
steady state
Remarks on result:
other: 34d BCF of ADONA at a concentration of 0.1 mg/L active ingredient was 0.094 ± 0.0071.
Remarks:
Conc.in environment / dose:0.1 mg/L active ingredient
Details on results:
- Mortality of test organisms: No mortality or other adverse effects/disease was observed in treated fish during the whole test period.
- Behavioral abnormalities: None
- Observations on body length and weight: Not reported
- Other biological observations: None
- Mortality and/or behavioral abnormalities of control: No mortality or adverse effects/disease were observed in control fish during the whole test period.
- Loss of test substance during test period: None, test substance was stable during the test period. Analysis of the actual concentrations were generally in agreement with the target concentrations (between 90 - 122%).

ADONA concentrations in fish can be found in Table 1. ADONA concentrations in the test medium can be found in Table 2. The BCF values for ADONA can be found in Table 3. The percentage of lipid in carp can be found in Table 4. The experimental conditions of the study can be found in Table A.

The uptake of ADONA by Cyprinus carpio was insignificant (BCF < 10, Table 3), so a depuration phase was not needed and the study was terminated after 34 days of exposure.
Reported statistics:
The fish did not concentrate ADONA, therefore, the BCF was calculated over the whole exposure period of 34 days. The steady state BCF was determined from the division of the mean concentration of test substance in fish by the mean concentration of test substance in water.

Any other information on results incl. tables

Table 1, Concentrations of ADONA in Carp

Day of Sampling

Measured Concentrations ¹

[mg/kg]

Control ²

0.1 ²

1.0 ²

1

0.00224

0.0116

0.0770

0.00219

0.0102

0.0986

 

0.00821

0.0872

 

0.0118

0.105

Mean

 

0.010

0.092

3

<0.002

0.0110

0.0969

<0.002

0.0128

0.113

 

0.0107

0.0814

 

0.00851

0.0821

Mean

 

0.011

0.093

7

<0.002

0.0103

0.0610

<0.002

0.00912

0.0379

 

0.0103

0.0607

 

0.0925

0.0377

Mean

 

0.010

0.049

10

<0.002

0.00465

0.0636

<0.002

0.00516

0.0651

 

0.00583

0.0659

 

0.00474

0.0627

Mean

 

0.0051

0.064

20

<0.002

0.00905

0.0800

<0.002

0.0127

0.0960

 

0.00886

0.0818

 

0.00847

0.0969

Mean

 

0.010

0.089

30

<0.002

0.00837

0.102

<0.002

0.0115

0.106

 

0.0114

0.115

 

0.0109

0.0808

Mean

 

0.011

0.10

34

<0.002

0.00856

0.0765

<0.002

0.0100

0.0658

 

0.0101

0.0897

 

0.00867

0.0746

Mean

 

0.0093

0.077

1) Concentrations for the active ingredient, corrected for the purity of the test substance (29.9%)

B) Exposure concentrations (mg/L active ingredient)

Table 2, Concentration of ADONA in test medium

Day of sampling

ConcentrationA

Target

[mg/L]

Analyzed

[mg/L]

Relative to

Target

[%]

-1

0

n.d.B

n.a.C

0.1

0.103

103

1

1.18

108

 

 

 

 

0

0

n.d.

n.a.

0.1

0.100

100

1

1.22

122

 

 

 

 

1

0

n.d.

n.a.

0.1

0.102

102

1

1.09

109

 

 

 

 

3

0

n.d.

n.a.

0.1

0.116

116

1

1.20

120

 

 

 

 

7

0

n.d.

n.a.

0.1

0.103

103

1

0.900

90

1D

0.171

17

 

 

 

 

10

0

n.d.

n.a.

0.1

0.0559

56

1

0.897

90

 

 

 

 

13

0

n.d.

n.a.

0.1

0.107

107

1

1.09

109

 

 

 

 

20

0

n.d.

n.a.

0.1

0.112

112

1

1.21

121

 

 

 

 

30

0

n.d.

n.a.

0.1

0.0992

99

1

1.13

113

 

 

 

 

34

0

n.d.

n.a.

0.1

0.107

107

1

1.20

120

A) Concentration for active ingredient, corrected for the purity of the test substance (29.9%)

B) Not determined

C) Not available

D) Extra sample

Table 3,  BCF value of ADONA

Day

Target Concentration ¹: 0.1 mg/L

 

Target Concentration ¹: 1.0 mg/L

Cw ²

[mg/L]

Cf ³

[mg/kg]

BCF

 

Cw ²

[mg/L]

Cf ³

[mg/kg]

BCF

 

1

0.10

0.010

0.10

 

1.1

0.092

0.084

3

0.12

0.011

0.093

 

1.2

0.093

0.078

7

0.10

0.010

0.094

 

0.90

0.049

0.055

10

0.056

0.0051

0.091

 

0.90

0.064

0.072

13

0.11

 

 

 

1.1

 

 

20

0.11

0.010

0.087

 

1.2

0.089

0.073

30

0.10

0.011

0.11

 

1.1

0.10

0.089

34

0.11

0.0093

0.087

 

1.2

0.077

0.064

1) Concentration for active ingredient, corrected for purity of test substance (29.9%)

2) Measured mean concentration of active ingredient in test medium

3) Measured mean concentration of active ingredient in fish

Table 4, Percentage of lipid in fish

Target Concentration ¹

[mg/L]

Weighed

Amount

[g]

Weight

vessel

[g]

Weight

vessel +

Lipid

[g]

Lipid

[g]

Lipid

[%]

Day 0

 

 

 

 

 

Control

8.79

99.78

100.1

0.32

4

Day 30

 

 

 

 

 

0.1

14.04

98.14

98.5

0.36

3

1.0

13.49

96.57

97.0

0.43

3

1) Concentration for active ingredient, corrected for purity of test substance (29.9%)

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Temperature variation ± 2 °C; Dissolved O2 concentration > 5 mg/L; Variation of actual test substance concentrations in water within ± 20% mean measured values; No mortality or adverse effects in either control or treated fish for whole test.
Conclusions:
The ADONA 34d mean BCF for 0.1 mg/L active ingredient concentration was 0.094 ± 0.0071 and for 1.0 mg/L active ingredient concentration was 0.074 ± 0.012.
Executive summary:

DONA is an acid that is expected to be deprotonated under conditions of the bioconcentration test and in the environment. ADONA is the ammonium salt of DONA. The detection method used in this test was specific for the DONA anion. Bioconcentration data for ADONA can therefore be used to assess DONA bioconcentration.

The mean BCF of ADONA at concentrations of 0.1 and 1.0 mg/L active ingredient for a 34 day uptake period were 0.094 ± 0.0071 and 0.074 ± 0.012, respectively. As the BCF for the uptake was < 10, a depuration phase was not needed. Exposure concentrations were based on nominal concentrations as analytical results showed that concentrations were within 90 - 122% during the test period. The lipid extraction performed on control fish at the start of the study showed 4% lipid. On day 30 of the uptake period, test concentration fish showed 3% lipid at both 0.1 and 1.0 mg/L active ingredient. There is no substantial risk for bioconcentration of DONA in fish.

Results Synopsis

Test Type: Flow through (based on data obtained using OECD 305 methodology)

34d BCF for 0.1 mg/L active ingredient: 0.094 ± 0.0071

34d BCF for 1.0 mg/L active ingredient: 0.074 ± 0.012

The 0.1 and 1.0 mg/L active ingredient concentrations correspond to nominal concentrations of 0.33 and 3.3 mg/L total product.