potassium (oxido-NNO-azoxy)cyclohexane; cyclohexylhydroxydiazene 1-oxide, potassium salt; [K-HDO]

Long-term toxicity to fish

Administrative data

Endpoint:

fish, juvenile growth test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Apr 21, 2005 to Mai 19, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 215 (Fish, Juvenile Growth Test)

Version / remarks:

adopted January 21, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexylhydroxydiazene 1-oxide, potassium salt

EC Number:

613-953-8

Cas Number:

66603-10-9

Molecular formula:

C6H11KN2O2

IUPAC Name:

Cyclohexylhydroxydiazene 1-oxide, potassium salt

Test material form:

solid: particulate/powder

Details on test material:

Test item: (N-Cyclohexyl-diazeniumdioxy)-potassium
IUPAC name: Cyclohexylhydroxydiazene 1-oxide, potassium salt
Chemical name: Cyclohexylhydroxydiazene 1-oxide, potassium salt; synonyma: (N-Cyclohexyl-diazeniumdioxy)-potassium, K-HDO, K-NCH, Xyligen K powder, Xyligen K
Molecular formula: C6 H11 K N2 O2
Molecular mass: 182.27

Specific details on test material used for the study:

Name: N-cyclohexyl-diazeniumdioxy-potassium
Test substance No.: 01/0069-6
Batch No.: W-87
CAS No.: 66603-10-9
Purity: 100%
Homogeneity: Homogeneous
Stability under storage conditions: The stability under storage conditions over the exposure period was guaranteed
IUPAC name: N-cyclohexyl-diazeniumdioxy-monohydrat potassium
Synonyms: K-HDO
Production date: Dec 2000 - Jan 2001
Physical state / Appearance: Solid, crystalline, white
Solubility in water: Soluble

The analytical examinations of the test substance were conducted at the Analytical Department of BASF Aktiengesellschaft

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

ANALYTICAL VERIFICATION:

- CALIBRATION PHASE: Samples of one test vessel per concentration group were taken one day before start of exposure and it was confirmed that the concentrations were within a range of ± 20% of the nominal concentrations. The analyses were not performed under GLP and are not reported in detail.

- EXPOSURE PERIOD: Samples were collected on day zero from each test vessel and subsequently at weekly intervals alternating from one test vessel per concentration group before the replacement of the stock solutions. These samples were analyzed for the content of the test substance.

Samples were taken from the middle of the test vessel using a beaker and were transported to the analytical laboratory in glass bottles. Retained samples were frozen and sent later to the analytical laboratory if necessary.

Test solutions

Vehicle:

yes

Remarks:

Non chlorinated charcoal filtered tap water (Frankenthal, Germany), mixed with deionized water.

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Three different stock solutions were prepared to provide the test concentrations with the appropriate amounts of active ingredient. Stock solution I (375 mg active ingredient / L dilution water) was used to supply the test concentration 3.3 mg/L, stock solution II (125 mg active ingredient / L dilution water) was used to supply the test concentration 1.1 mg/L and stock solution III (50 mg active ingredient / L dilution water) was used to supply the test concentrations 0.33, 0.11 and 0.033 mg/L.
A stock solution of 11 g/L test substance in the dilution water was prepared. From this stock solution aliquots of 375, 125 and 50 ml were diluted with 10 L dilution water, respectively, to prepare the stock solutions I, II and IlI to be used in the flow through system. The stock solutions are prepared in glass bottles and mixed by shaking. At the end of the test only half of the volumes given above were prepared.
After the first preparation of the stock solutions, the contents of the test substance were determined analytically. The analyses confirmed the complete solution of the entire test substance. The stock solutions were "true" solutions. Therefore, no further analyses of the stock solutions were conducted.
Throughout the exposure period the stock solutions are continuously diluted with dilution water at constant rates and the mixture was flowing into the test vessels. Renewal of the stock solutions was done in weekly intervals.
Details of the dilution steps are presented in the table below.

Test organisms

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: zebrafish
- Strain: Danio rerio
- Source: testing facility
- Age at study initiation (mean and range, SD): juveniles
- Length at study initiation (length definition, mean, range and SD): The mean body length (from the tip of the snout to the distal end of the caudal fin) for all fish assigned to the test groups at start of exposure ± standard deviation was 20.5 ± 1.0 mm
- Weight at study initiation (mean and range, SD): The overall mean weight at start of exposure ± standard deviation was 65.0 ± 5.7 mg. The lowest value was 56 mg (86% of the mean value), the highest value was 71 mg (109% of the mean value). Thus the body weight of the fish at start of exposure was sufficiently homogeneous and was well within the range of ± 25%, which is acceptable according to the test guideline.
- Feeding during test: During the exposure period, the juvenile fish were offered newly hatched brine shrimp larvae (Artemia naupli)
- Amount and frequency: The quantity of feeding (dry weight of artemia) was adjusted to 4% of the mean body weight of the fish at start of exposure for the first two weeks and to the bodyweight of the intermediate weighing from day 13 on. The daily ration was divided into two equal portions and given to the fish in two portions per day, separated by at least five hours. On weekends and holidays, the interval between the two feedings was reduced.
Shorter intervals were used also on the day on which the exposure was started for technical reasons and on the day before end of exposure, since the feeding should be ended one day before sacrifice. No food was applied for approx. 24 hours before the termination of exposure on day 28.
- Test vessels: glass aquaria (inner dimensions: 39 cm long, 24 cm wide, 30 cm high) with an overflow located 26 cm above the bottom. The overflow was covered with a stainless-steel gauze to prevent the juvenile fish from escaping. Each aquarium was maintained with a constant water volume of approximately 25 litres.

ACCLIMATION
All animals used for the study were derived from the same batch of fish. A sufficient number of fish was acclimated to the dilution water with a hardness of 140 mg/L CaCO3 for 9 days before the exposure was started. No mortality was observed in this group during acclimation. All test animals were observed for their health state during insertion into the test vessels. No signs for sickness, injuries or abnormalities were observed.

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

28 d

Post exposure observation period:

Not required

Test conditions

Hardness:

1.42 mmol/l (1 mmol corresponds to approx. 100 mg CaCO3/L)

Test temperature:

24 °C

pH:

7.9

Dissolved oxygen:

8.3 mg/L

Conductivity:

334 µSi

Nominal and measured concentrations:

Nominal concentrations were: 0.0 mg/L (Control), 0.033, 0.11, 0.33, 1.1 and 3.3 mg/L. The concentrations were based on the content of active ingredient N-cyclohexyl diazenium dioxy monohydrate potassium in the test substance (100%).
The mean analytically determined concentration values of the test substance in the test water were in good agreement with the nominal concentrations in the 3 lower concentrations (88.9 - 100.8%). In the 1.1 mg/L test group the mean measured concentration was 74.3% of the nominal concentration, in the 3.3 mg/L test group it was 53.2% of the nominal value.
However, in all test groups all measured concentrations were in the range of ± 20% of the mean measured concentration, with exception of the start value of the lowest concentration, which was 123.5% of the mean value. Thus, constant concentrations were maintained during the test and the results should be based on the mean measured concentration. The effect concentrations based on the mean of the analytically determined concentrations are reported in addition to the results based on nominal concentrations.
The mean measured concentrations were (% of nominal):
0.033 mg/L = 100.8%, 0.11 mg/L = 88.9%; 0.33 mg/L = 97.0%; 1.1 mg/L = 74.3% and 3.3 mg/L = 53.2% of the nominal values, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria (inner dimensions: 39 cm long, 24 cm wide, 30 cm high) with an overflow located 26 cm above the bottom. The overflow was covered with a stainless-steel gauze to prevent the juvenile fish from escaping. Each aquarium was maintained with a constant water volume of approximately 25 litres.
- Aeration: The aquaria were not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): The flow rates of the test solutions and the water of the control were 5.2 L/hour/test vessel, respectively. Taking into account the water volume of the aquaria, the theoretical exchange rate of the water contents was approximately 5 fold per 24 hours.
- No. of organisms per vessel: Each test group and the control group consisted of one aquarium with 20 juvenile fish.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon: 1.45 mg/L
- Total suspended solids (mg/L): 9
- Metals: free of heavy metals
- Pesticides: free of pesticides
- Chlorine: none-chlorinated
- Alkalinity: pH = 7.9
- Culture medium different from test medium: The fish were not treated with any medication and kept in general under the same conditions as during the test with exception of the water hardness, which was adjusted to 100 mg/L CaCO3.
- Intervals of water quality measurement: see below

OTHER TEST CONDITIONS
- Adjustment of pH & oxygen: The pH and the content of oxygen dissolved in the water were routinely determined twice weekly in all test vessels, also once during the exposure period from the dilution water of the water supply.
- Photoperiod: The aquaria were exposed to dim light at a light cycle of 16 hours light and 8 hours darkness, which was automatically maintained.
- Physico-chemical parameters: The total hardness of the test water from the control and the highest test concentration were determined at start and end of exposure by titration with EDTA. The acid capacity of the test water from the control and the highest test concentration were determined at start and end of exposure by titration with HCL according to DIN (German Industrial Standard) No. 38409. Measurements of the following parameter were performed once during the exposure period in the water supply of the control group: conductivity, acid capacity, temperature, total organic carbon, total suspended solids, total hardness.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Measured and / or determined biological parameters were the mortality of the juvenile fish, toxic signs (symptoms), the weight (wet weight) after 14 days (For technical reasons the intermediate weighing was performed already after 13 days. This change has no effect on the evaluation of the parameter and the results of the study) of exposure and the weight (wet weight) as well as the total length (from tip of the snout to the end of the caudal fin) of surviving fish at the end of exposure.

RANGE-FINDING STUDY
- Test concentrations:
According to the OECD Guideline 5 concentrations spaced by a constant factor and a control without test substance should be tested. The test should include at least one concentration with and another without an adverse effect on the test organisms.
The test concentrations were selected on the basis of preliminary tests, which indicated mortality at 10.0 mg/l within a test duration of 4 days. The following concentrations spaced by a factor of √10 were selected for this study: 0.0 (dilution water control) / 0.033 / 0.11 / 0.33 / 1.1 and 3.3 mg/L. Since the purity of the test substance is 100 %, the concentrations based on the test substance and on active ingredient are the same.
- Results used to determine the conditions for the definitive study: Mortality at 10.0 mg/l within a test duration of 4 days

Reference substance (positive control):

not specified

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.33 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival and growth rate

Effect concentrations 2

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

0.29 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival and growth rate

Effect concentrations 3

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival and growth rate

Effect concentrations 4

Key result

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

0.74 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: survival and growth rate

Details on results:

- Fish weights (individual and mean values) on day 0, 14 (if measured) and 28: see table below
- Tank-average or pseudo-specific growth rates for periods 0-28 or 0-14 and 0-28 (if possible): see table below
- External abnormalities and abnormal behaviour: over the exposure period no toxic signs and no abnormalities in the control and in the surviving fish of the concentration groups were observed.
- Other biological observations: none observed
- Effect concentrations exceeding solubility of substance in test medium: not applicable
- Incidents in the course of the test which might have influenced the results: none

Results with reference substance (positive control):

No reference substance used.

Reported statistics and error estimates:

Body weight, Length, R3: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means
Mortality: A comparison of each group with the control group was performed using Fisher's exact test (one-sided)

Any other information on results incl. tables

Survival rate

Test group	Nominal concentration (mg/L)	Mean analytical concentration (mg/L)	Survivors at end of study	Percentage of survivors related to individuals at study start
0	0 (control)	n.d.	20	100 %
1	0.033	0.0411	20	100 %
2	0.11	0.100	20	100 %
3	0.33	0.334	20	100 %
4	1.1	0.683	6	30 %
5	3.3	1.787	0	0 %

 

Mean body lengths of the concentration groups and the control group at the end of the study were:

Test group	Nominal concentration (mg/L)	Mean analytical concentration (mg/L)	Mean body length of individual fish (mm)
0	0 (control)	n.d.	30.4
1	0.033	0.0411	30.4
2	0.11	0.100	30.0
3	0.33	0.334	30.0
4	1.1	0.683	25.3
5	3.3	1.787	-

- all individuals dead

Mean wet weights of the groups and the control group at the end of exposure were:

Test group	Nominal concentration (mg/L)	Mean analytical concentration (mg/L)	Mean weight of individual fish (mg)
0	0 (control)	n.d.	218.5
1	0.033	0.0411	220.4
2	0.11	0.100	216.4
3	0.33	0.334	217.1
4	1.1	0.683	180.2 (p < 0.05)
5	3.3	1.787	-

- all individuals dead

Tank / “Pseudo”-specific growth rates of the exposure groups in comparison to the control group at day 13 and at the end of exposure were:

Test group	Nominal concentration (mg/L)	Mean analytical concentration (mg/L)	Mean of “pseudo”-specific growth rate R3^1)			% of control			Statistical significance
			Days			Days
			0 - 13	13 – 28	- 28	0 - 13	13 – 28	0 - 28
0	0 (control)	n.d.	4.695	3.955	4.298	100	100	100
1	0.033	0.0411	4.629	4.098	4.345	98.6	103.6	101.1	None
2	0.11	0.100	4.604	4.131	4.350	98.1	104.5	101.2	None
3	0.33	0.334	4.854	3.669	4.219	103.4	92.8	98.2	None
4	1.1	0.683	1.890^2)	4.963^2)	3.536^2)	40.3^2)	125.5^2)	82.3^2)	Yes
5	3.3	1.787	-	-	-	-	-	-	-

¹⁾The mean values of R3 are similar to the R2-values (tank-average specific growth rate)

²⁾Mortality rate in this test group > 10 % 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

- the mortality in the control group was 0 %
- the mean body weight increase in the control group was > 50 % of the initial value
- the dissolved oxygen content was > 60 % of the air saturation value throughout the test
- the water temperature in all test vessels did not differ by more than 1 °C

In conclusion, the study was considered to be valid.

Under the conditions of this study, the overall NOEC (no observed effect concentration) was 0.33 mg/l (nominal concentration) and 0.29 mg/l (based on the mean analytically determined concentrations) and the lowest concentration with effects (LOEC) was 1.1 mg/l (nominal concentration) and 0.74 mg/l (based on the mean analytically determined concentrations).

Executive summary:

A test on effects on growth of N-Cyclohexyldiazeniumdioxy-potassium on juvenile zebra fish (Danio rerio) was conducted by the Laboratory for Wildlife and Fish Toxicology of Experimental Toxicology and Ecology, BASF AG, Ludwigshafen, Germany for a period of 28 days following the OECD guideline for Testing of Chemicals No. 215, adopted January 2000 “Fish, Juvenile Growth Test”.

The study was performed under flow-through conditions with 5 concentrations of the test substance and a dilution water control. The temperature was maintained generally at 24 °C. the dilution water was none-chlorinated drinking water obtained from the municipal water works mixed with deionised water to achieve a hardness of 1.4 mmol/L (149 mg/L CaCO₃)

The test concentrations were selected on the basis of preliminary tests, which indicated mortality at 10.0 mg/L within a test duration of 4 days. The following concentrations spaced by a factor of √10 were selected for this study: 0.0 (dilution water control) / 0.033 / 0.11 / 0.33 / 1.1 and 3.3 mg/L. Since the purity of the test substance is 100 %, the concentrations based on the test substance and on active ingredient are the same.

 

Survival:

In the control and the concentration groups up to 0.33 mg/L all fish survived until sacrifice. In the highest concentration group (3.3 mg/L), all fish died during the first day of exposure. In the concentration group 1.1 mg/L the survival rate was 30 %. Mortalities were observed from days 1 – 14 after start of exposure.

In conclusion, the NOEC for survival is 0.33 mg/L (nominal concentrations) and 0.29 mg/L based on the mean analytically determined concentrations. The LOEC is 1.1 mg/L (nominal concentration) and 0.74 mg/L based on the mean analytically determined concentrations.

 

External abnormalities and abnormal behaviour:

Over the exposure period, no toxic signs and no abnormalities in the control and in the surviving fish of the concentration groups were observed.

Growth rate:

In comparison to the control group the growth rate was statistically significantly reduced in the surviving animals of the concentration group 1.1 mg/L after 14 days (For technical reasons the intermediate weighing was performed already alter 13 days). No effects on the growth rate were detected in the lower concentration groups 0.033, 0.11 and 0.33 mg/L.

For technical reasons the intermediate weighing was performed already after 13 days. This change has no effect on the evaluation of the parameter and the results of the study.

Thus, the NOEC for the impairment of the growth rate was 0.33 mg/L (nominal concentration) and 0.29 mg/L (based on the mean analytically determined concentrations). The LOEC for the impairment of the growth was 1.1 mg/L (nominal concentration) and 0.74 mg/L (based on the mean analytically determined concentrations).

 

In conclusion, under the conditions of this study, the overall NOEC (no observed effect concentration) was 0.33 mg/L (nominal concentration) and 0.29 mg/L (based on the mean analytically determined concentrations) and the lowest concentration with effects (LOEC) was 1.1 mg/L (nominal concentration) and 0.74 mg/L (based on the mean analytically determined concentrations).