Copper, diammine[5,5'-bi-1H-tetrazolato(2-)-kN1, kN1']

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:

June - August 2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Report in Japanese. NO GLP certificate available in the report (reliability guarantee present). Iuclid informations according to english translation of the report.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 305 (Bioconcentration: Flow-through Fish Test)

Version / remarks:

Testing methods:
a)“In Vivo Bioconcentration Testing of Chemical Substances in Fish” stipulated in “Testing Methods for New Chemical Substances, etc.” (PMDA Notification No. 8, of 31 March, 2011; MIB Notification No. 6 of 29 March, 2011; Health and Chemicals Notification No. 110331010)
b)OECD Guidelines for Testing of Chemicals, No.305, June 14, 1996, "Bioconcentration: Flow-through Fish Test"

Deviations:

not specified

Remarks:

Test material is sligthly soluble and exposure was performed by water and not dietary route

GLP compliance:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Autoliv, Inc, Aichi Office
- lot/batch : IVM210-62

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a dark indoor location
- Stability under test conditions: The infra-red absorption spectrum measured by the testing facility was confirmed as being consistent with that provided by the test sponsor. In addition, the stability of the test material under the storage conditions was confirmed by comparing the infra-red absorption spectra before the start and after completion of testing
- Solubility: not determined, slightly soluble (visual)

Details on sampling:

- Sampling intervals/frequency for test organisms: day 2- 8 - 15- 22-28
- Sampling intervals/frequency for test medium samples: day 1- 2- 8 - 15- 22-28
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Because of the low solubility of the test material in general-purpose solvents and its behavior in water (liberation of 5,5'-bi-1H-tetrazole, copper an ammonia) , analysis of CuDABT itself was not possible in water and fish. Thus, the 5,5'-bi-1H-tetrazole composing the tested substance was the analysis subject. Furthermore, it was difficult to quantify copper due to the interference of blank constituents included in the analysis samples, therefore the concentration was checked qualitatively rather than by quantitative analysis. The analysis of the 5,5'-bi-1H-tetrazole in the test water as well as the test fish was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS).
Analysis Sample Pre-treatment Methodsin Test WaterSee Flow (schema 1 attached document)
In fish samplesSee Flow (schema 2 attached document)

Vehicle:

yes

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

The test material was ground with rock sugar and sodium polyacrylate and dissolved in ion-exchange water containing polyvinyl alcohol. The adapted volume of ion exchange water was added to prepare a stock solution at 100 mg/L and 32.0 mg/L . Adjunction controlsRock sugar, sodium polyacrylate, and polyvinyl alcohol were dissolved in ion-exchange water, and stock solutions prepared with respective concentrations of 640, 3 200, and 12 800 mg/L.

Test organisms (species):

Cyprinus carpio

Details on test organisms:

TEST ORGANISM
- Common name: Carp
- Source: Chemical evaluation and research institute , Japan, Kurumr Office
- Age at study initiation : Yearling fish
- Length at study initiation : 6.8- 8.5 cm
- Lipid content at test initiation : 3.64 %
- Health status:
- Description of housing/holding area: - Feeding during test - Food type: Type Blended feed for raising of carpProtein content 43.0% or moreFat content 3.0% or more - Feeding of an amount corresponding to about 3% of the test fish body weight was performed 2 times/day (these were combined into 1 time on holidays). However, feeding was stopped 24 hours before sampling of the test fish.

ACCLIMATION
Acclimation was performed under the following conditions after having placed in a aquacultural bath of OTC (oxy-tetracycline hydrochloride Kawasaki
Kagaku) and sodium chloride (The Salt Industry Center of Japan). PeriodAcclimated for 14 days in dechlorinated tap water after raising for 31 days in groundwater. Water Temperature Less than 25±2°C- Health during acclimation (any mortality observed):The overall mortality rate during the acclimation period was
less than 5%.

Route of exposure:

aqueous

Test type:

flow-through

Water / sediment media type:

other: Dechlorinated tap water

Total exposure / uptake duration:

ca. 28 d

Total depuration duration:

ca. 0 d

Hardness:

No measured

Test temperature:

24.4 - 24.9

pH:

7.8 - 8.0

Dissolved oxygen:

7.7 - 8.1 mg/L

TOC:

Not measured

Salinity:

NA

Conductivity:

Not measured

Details on test conditions:

TEST SYSTEM
- Test vessel: 70L capacity glass water tank
- Aeration: continuous
- Renewal rate of test solution (frequency/flow rate): 1155L /day suplied at a proportion of stock solution 2 mL/min and test water 800 mL/min
- No. of organisms per vessel: 28 for each test concentration, 12 for controls
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: Not specified

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

PARAMETERS (see table attached document)
- Intervals of water quality measurement: once during the test

OTHER TEST CONDITIONS
- Photoperiod: 14 hours day/ 10 hours night
- Light intensity: not specified, white fluorescent lamps

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: 8.00 mg/L
- Results used to determine the conditions for the definitive study: no mortality at 8.00 mg/L

Nominal and measured concentrations:

Nominal concentrations: 80 µg /L8 µg /LMeasured concentrations: see tables on attached document

Reference substance (positive control):

not specified

Details on estimation of bioconcentration:

The bioconcentration factor (BCF) was calculated according to the following formula:
a)Calculation of the Mean Tested Substance Concentration in Test Water for Bioconcentration Factor Calculation
Cw = (Cw(n-1) + Cw(n))/ 2 (1st Test Fish Analysis)
Cw = (-2Cw(n-1) + Cw(n))/ 3 (2nd Test Fish Analysis onward)
Cw:Mean tested substance concentration in test water for bioconcentration factor calculation (µg/L)
Cw(n):nth test water analysis of tested substance concentration derived simultaneously with the test fish analysis (µg/L)

b)Bioconcentration Factor Calculation
BCF = Cf / Cw
BCF:Bioconcentration factor
Cf:Tested substance concentration in test fish (value with FB subtracted) (ng/g)
Cw:Mean tested substance concentration in test water for bioconcentration factor calculation (µg/L)
FB:Mean value of tested substance in test fish before start and after end of experiment in the control zone or baseline (blank) concentration of tested substance (ng/g)

c)Mean bioconcentration factor for m times
BCFm = (BCFa + BCFb)/ n
BCFm:Mean bioconcentration factor of the time m (No. of groups 2 (a,b))
BCFa,b:Bioconcentration factor for each group at the time mn:No. of analysis groups analysed at the time mHowever, the mean value of the bioconcentration factors was not derived for measurement days with non-detection.

Lipid content:

ca. 3.64 %

Time point:

start of exposure

Lipid content:

ca. 3.52 %

Time point:

end of exposure

Key result

Conc. / dose:

ca. 80 µg/L

Temp.:

>= 24.5 - <= 24.8 °C

pH:

8

Type:

BCF

Value:

< 1.2 dimensionless

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Key result

Conc. / dose:

ca. 8 µg/L

Temp.:

> 24.6 - < 24.9 °C

pH:

7.9

Type:

BCF

Value:

< 12 dimensionless

Basis:

whole body w.w.

Time of plateau:

28 d

Calculation basis:

steady state

Details on results:

- Mortality of test organisms:0
- Behavioural abnormalities: no
- Mortality and/or behavioural abnormalities of control: 0
- Loss of test material during test period: < 20%

Regarding copper Bioconcentration

The  copper  in  the  tested  material  was  not  quantitatively  analysed  through  interference  of  the  blank constituents contained in the analysis samples. Here, the presence of copper concentration in the tested material using test fish refined samples was confirmed in the 1st concentration and 2nd concentration after day 28, as well as in the control, after experiment completion through inductively coupled plasma atomic emission spectroscopy (ICP-AES).

The  table  below  shows  the  emission  intensities  of  the  various  analysis  samples.  Upon  calculating  the bioconcentration factors of the bioconcentration factors of the test fish samples of the 1st concentration (80 µg/L) , sample a was 16× and sample b was 20×. For the 2nd concentration (8 µg/L), it was determined that  the  test  material  was  not  detected  as  the  emission  intensity  was  on  the  same  order  as  in  the control .The concentration of copper in the test substance was thought to be low from the above results. Furthermore, the test material concentration in the test water for bioconcentration factor calculation used the set concentration.

Emission Intensity of the Various Analysis Samples (Units cps)

Sample	Emission Intensity
160 µg/L Standard Solution	2131.8
Post-28 Day 1st Concentration Sample a	2200.4
Post-28 Day 1st Concentration Sample b	2325.0
160 µg/L Standard Solution	2150.3
Post-28 Day 2nd Concentration Zone Sample a	1549.0
Post-28 Day 2nd Concentration Zone Sample b	1656.0
160 µg/L Standard Solution	2208.0
Post-experiment completion Control Sample a	1519.5
Post-experiment completion Control Sample b	1959.4

Validity criteria fulfilled:

yes

Conclusions:

Because of the low solubility of the test material and its behavior in water (release of 5,5'-bi-1H-tetrazole, copper an ammonia), analysis of CuDABT itself was not possible in water and fish. Then, the 5,5'-bi-1H-tetrazole composing the test material was the analysis subject. Furthermore, it was difficult to quantify copper due to the interference of blank constituents included in the analysis samples, therefore the concentration was checked qualitatively rather than by quantitative analysis. The analysis of the 5,5'-bi-1H-tetrazole in the test water as well as the test fish was performed by liquid chromatography / tandem mass spectrometry (LC-MS/MS). Based on the 5,5'-bi-1H-tetrazole analysis, no bioconcentration was measured in fish at 80 µ/L and 8 µg/L after 28 days of exposure.Based on copper determination, the bioconcentration factors of the test fish samples of the 1st concentration, was 16× and 20× (sample a and sample b). For the 2nd concentration , it was determined that the test material was not detected, as the emission intensity was on the same order as in control condition .

Executive summary:

CuDABT bioconcentration test was performed using flow trought exposure on Carp, acording to the following test methods

a)    “In Vivo Bioconcentration Testing of Chemical Substances in Fish” stipulated in “Testing Methods for New Chemical Substances, etc.” (PMDA Notification No. 8, of 31 March, 2011; MIB Notification No. 6 of 29 March, 2011; Health and Chemicals Notification No. 110331010)

b)    OECD Guidelines for Testing of Chemicals, No.305, June 14, 1996, "Bioconcentration: Flow-through Fish Test"

Test material stock solutions were prepared as follows:

The  test  material  was  ground  with  rock  sugar  and  sodium  polyacrylate  and  dissolved  in  ion-exchange water  containing  polyvinyl  alcohol.  The  adapted  volume  of  ion  exchange  water  was  added  to  prepare stock solutions at 100 mg/L and 32 mg/L. Stock solution were added to flow through system to reach test concentration at 80 µg/L and 8 µg/L.

Because of the low solubility and the behavior of the test material in water ( release of 5,5'-bi-1H-tetrazole ammonia  and  copper),  analysis  of  CuDABT  itself  was  not  possible  in  water  and  fish.  Then,  the  5,5'-bi-1H-tetrazole composing the tested substance was the analysis subject. Furthermore, it was difficult to quantify copper due to the interference of blank constituents included in the analysis samples, therefore the concentration was checked qualitatively rather than by quantitative analysis. The analysis of the 5,5'-bi-1H-tetrazole in the test water as well as the test fish was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS).

Bioconcentration factors for 5,5'-bi-1H-tetrazole were not determinable ( lower than 1.2 for 80µg/L and lower than 12 for 8 µg/L).

For copper, the bioconcentration factors of the test fish samples of the concentration at 80 µg/L was 16× and 20× (sample a and sample b). For the concentration at 8µg/L, it was determined that the tested substance was not detected as the emission intensity was on the same order as the control condition.

Description of key information

Because of the low solubility of the test material and its behavior in water (release of 5,5'-bi-1H-tetrazole, copper an ammonia), analysis of CuDABT itself was not possible in water and fish. Then,  the 5,5'-bi-1H-tetrazole composing the test material was the analysis subject. Furthermore, it was difficult to quantify copper due to the interference of blank constituents included in the analysis samples, therefore the concentration was checked qualitatively rather than by quantitative analysis. The analysis of the 5,5'-bi-1H-tetrazole in the test water as well as the test fish was performed by liquid chromatography / tandem mass spectrometry (LC-MS/MS). Based on the 5,5'-bi-1H-tetrazole analysis, no bioconcentration was measured in fish at 80 µ/L and 8 µg/L after 28 days of exposure ( lower than 1.2 for 80µg/L and lower than 12 for 8 µg/L).Based on copper determination, thebioconcentration factors of the test fish samples of the 1st concentration, was 16× and  20× (sample a and sample b). For the 2nd concentration , it was determined that the test material was not detected, as the emission intensity was on the same order as in control condition .