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

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

adopted September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Version / remarks:

April 1996

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:

liquid

Details on test material:

Xyligen 30F is a 30% aqueous solution of K-HDO.

Specific details on test material used for the study:

Name: Xyligen K 30 F
Batch No.: U8456
Purity: 31.4 % in water
Certificate of Analysis Date: July 17, 2001
Aggregate State at RT: liquid
Colour: yellowish-brown
Solubility: in water: miscible up to 10 g/L
Stability: stability during the test period was guaranteed
Storage: in original container, at room temperature, in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

SAMPLING:
One sample from the freshly prepared stock solutions and duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the first treatment period (day 0) and at a treatment period in the second and third week (days 10 and 17).
For the determination of the stability of the test item under test conditions respectively the maintenance of the test item concentrations during the test period at three dates (days 3, 12 and 19) a sufficient volume from the freshly prepared test media of all test concentrations and the control were incubated under the same conditions as the test itself, however without food and Daphnia.
One of these three stability control treatments last for 72 hours (weekend), two for 48 hours, corresponding to the different test medium renewal periods.

STORAGE OF THE SAMPLES:
All samples of the nominal test concentrations of 0.39, 3.13 and 50.0 mg/L were analysed directly after sampling. All other test concentrations were stored in a refrigerator. After delivery of the final test report all samples will be discarded.

ANALYSES:
The concentrations of the test item were measured in all duplicate test medium samples from the lowest (0.39 mg/L), a middle (3.13 mg/L) and the highest test concentration (50.0 mg/L). From the control samples, only one of the duplicate samples was analysed from each sampling date.

Test solutions

Vehicle:

yes

Remarks:

water

Details on test solutions:

Test Concentrations: 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25.0 and 50.0 mg test item/L, and a control

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
Species: Daphnia magna (Straus), clone 5
Age at Test Start: 6.5 - 20.5 hours old
Sex: Female
Origin: supplied 1997 by the "Umweltbundesamt, lnstitut für Wasser-,Boden-, und Lufthygiene", Berlin, Germany.
Breeding Conditions: The Daphnia are bred in the laboratories of IBACON under similar temperature and light conditions as in the test, and in the same kind of reconstituted water used in the test. The test organisms were not first brood progeny.
Acclimatisation: under test conditions

TEST UNITS
Type and Size: 100 mL glass beakers containing 80 mL test medium
Identification: Each test unit was uniquely identified with study number, treatment and replicate number.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

not required

Test conditions

Hardness:

Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3
Alkalinity: 0.9 mmol/L

Test temperature:

21 °C

pH:

pH 7.7 to 8.5

Dissolved oxygen:

at least 8.1 mg/L or higher

Salinity:

not mentioned in the test report

Conductivity:

not mentioned in the test report

Nominal and measured concentrations:

During the test period at the test item concentrations of nominal 3.13 mg/L and 50 mg/L concentrations were measured in the range from 73 % to 108 % of the nominal values. In the mean 94 % and 103 % of nominal were determined. At the lowest test concentration of 0.39 mg/L in the mean 65 % of nominal was determined. However, this test concentration is below the limit of quantification. Under the test conditions the test item was sufficiently stable during the test medium renewal periods of 48 and 72 hours. Since the determined concentrations at the nominal test concentrations of 3.13 and 50 mg/L were well within the range of 80 % to 120 %, all reported results are related to nominal concentrations of the test item.

Details on test conditions:

TEST SYSTEM
- Type and Size: 100 mL glass beakers containing 80 mL test medium
- Identification: Each test unit was uniquely identified with study number, treatment and replicate number.
- Type of flow-through (e.g. peristaltic or proportional diluter): semi-static test
- Renewal rate of test solution (frequency/flow rate): the test media of all test concentrations and of the control were renewed on days 3, 5, 7, 9, 12, 14, 16 and 19 of the exposure period (every Monday, Wednesday and Friday). By that, a total of 9 treatments were performed. At these dates the test animals were carefully transferred by glass tubes from the old test vessels into the freshly prepared test media of the corresponding concentrations.
- No. of organisms per vessel: 10 Daphnia per control and test concentration, each in 80 mL test medium

TEST CONDITIONS
Surrounding Type: controlled environment room
pH-Value of Test Water: pH 7.7 - 8.5
Light Intensity: between 390 and 450 lux
Light Regime: 16 h light: 8 h dark

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water (Elendt "M4")
- Alkalinity: 0.9 mmol/L
- Culture medium different from test medium: no
- Water Hardness: 2.5 mmol/L (= 250 mg/L) as CaCO3

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Mortality of Adults and Number of Young: The mortality of the test animals and the number of young were recorded each day. Dead animals and offspring were removed at the same times.
- Measurements of pH, Dissolved Oxygen and Water Temperature: The pH-values and dissolved oxygen concentration in the control and all test concentrations were measured at the start and end of each treatment period. The water temperature was measured in one control beaker at the start and end of each treatment period.
- Behaviour of the Test Item in Test Water: The behaviour of the test item in test water was determined at each test medium renewal period in the freshly prepared and old test media of all test concentrations.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Test concentrations: 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25.0 and 50.0 mg test item/L. Additionally, a control was tested in parallel (test water without addition of the test item).

Reference substance (positive control):

not required

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.13 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

9.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Details on results:

see tables below

Results with reference substance (positive control):

not applicable

Any other information on results incl. tables

Number of surviving adult daphnia exposed to test item on day 21:

	Nominal concentration of Xyligen 30 F (mg/L)
Control	0.39	0.78	1.56	3.13	6.25	12.5	25.0	50.0
100 %	90 %	80 %	90 %	100 %	100 %	100 %	50 %	10 %

 

Total number of alive, young daphnia reproduced by all adults (cumulative values) in % of control on day 21

	Nominal concentration of Xyligen 30 F (mg/L)
Control	0.39	0.78	1.56	3.13	6.25	12.5	25.0	50.0
100 %	93.7 %	81.6 %	89.6 %	86.4 %	82.4 %	50.0 %	2.4 %	0.0 %

Number of alive offspring reproduced per surviving adult within 21 days of exposure (mean reproduction rate)

	Nominal concentration of Xyligen 30 F (mg/L)
Control	0.39	0.78	1.56	3.13	6.25	12.5	25.0	50.0
106.0	104.3	96.0	102.1	91.6	87.3	52.0	3.0	0.0

Toxic Effects: At the LOEC the mean reproduction rate of the surviving test animals was statistically significant lower than in the control. The survival rate of Daphnia magna after 21 days was significantly reduced first at the nominal test concentration of 25 mg test item/L.

 

Other effects: With the exception of the reported mortality and the reduced reproduction rates, no particular signs of intoxication were observed at the test animals during the test.

 

Results of controls: In the control, all the daphnia survived until the end of the test. The first young daphnia released from their parent animals were recorded in the control group at the observation on day 8. The mean reproduction rate for each individual daphnia, which survived until the end of the test in the control group was 106 + 13.3 (mean + SD).

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Taking into account the survival rates and the reproduction rates of the test animals, the highest concentration of Xyligen 30 F tested without toxic effects after the exposure period of 21 days (21-days NOEC) was 1.56 mg test item/L. The lowest concentration tested with toxic effects (21-days LOEC) was determined to be 3.13 mg test item/L due to the statistically significantly reduced mean reproduction rate of Daphnia magna at this test concentration.

Executive summary:

Survival of Adults:

In the control, all Daphnia survived until the end of the test. At the test concentrations of nominal 0.39 and 1.56 mg test item/L 90 % of the Daphnia survived and at the test concentration of 0.78 mg test item/L a survival rate of 80 % was determined. At the three next higher test concentrations of nominal 3.13 to 12.5 mg/L all Daphnia survived until the end of the test. At the nominal concentration of 25 mg test item/L the mortality rate increased to 50 % and at the highest test concentration of nominal 50 mg test item/L, one Daphnia survived until test end.

Thus, the survival rate of Daphnia magna after 21 days was significantly (> 20 %) reduced first at the nominal test concentration of 25 mg test item/L.

 

Reproduction Rate:

The first young Daphnia released from their parent animals were recorded in the control and the nominal test concentrations up to 6.25 mg/Lat the observation on day 8. At the test concentration of 12.5 mg test item/L first offspring was observed on day 10 and at the test concentration of 25 mg/L on day 14 the first offspring was determined. At the highest concentration tested (50 mg test item/L), no offspring was reproduced.

Reproduction rates were calculated for each individual Daphnia, which survived until the end of the test. The mean reproduction rate of those Daphnia in the control was 106.0 ± 13.3 (mean ± SD) alive offspring per adult. According to the results of a Williams-test (one-sided, a.= 0.05) no significant toxic effect of the test item on the mean reproduction rate was determined up to and including the nominal test concentration of 1.56 mg test item/L. First at the nominal concentration of 3.13 mg test item/L, the mean reproduction rate of surviving Daphnia was statistically significantly reduced to in the mean 86.4 % alive offspring compared to the control. The EC₅₀of the reproduction rate was determined to be 9.7 mg test item/L, with 95 % confidence limits from 4.3 to 54.1 mg/L.

Signs of Intoxication: With exception of the reported mortality and the reduced reproduction rates, no particular signs of intoxication were observed at the test animals during the test.