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

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

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 of test substance: N-cyclohexyl-diazeniumdioxy-potassium
Other names: Xyligen 30 F, Xyligen K 30 F U 8456
Batch number: U 8456
Substance number: 01/0069-2
Date of production: 10 July 2001
Aggregate state: Liquid
Density [g/ml]: 1.13 at 20°C
Water-solubility: miscible in any ratio
Colour: yellowish-brown
Purity of the test substance: 31.4% K-HDO in aqueous solution
Homogeneity: Homogenous
Instability against: Acid
Stability: The stability of the test substance is guaranteed until July 2002
Storage conditions: ambient (RT)
Expiry date: 10 July 2002

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples for analysis of the test concentrations were taken at Jan 29, 2002 and Feb 01, 2002. The results of the Concentration control of "Xyligen K 30 F" in "OECD-Medium Teil 9 einfach konzentriert" are presented in the table below.

Test solutions

Vehicle:

no

Details on test solutions:

The test substance was tested in the concentration range between 100 and 0.39 mg/L. The dilution factor was 2.
The test substance was stirred in demineralized water for about 20 min at approximately 20 ± 2 °C.
The nominal concentration of the stock solution was 125 mg/L.
By diluting this stock solution, the following nominal concentrations (mg/L) were prepared:
Control 100 50 25 12.5 6.25 3.13 1.56 0.78

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Desmodesmus subspicatus Chodat SAG 86.81
- Source/supplier: SAG (collection of algal cultures, Göttingen)
- Method of cultivation: liquid culture in BASF AG, seed culture was taken to inoculate a pre-culture
- Control group: yes
- Initial cell concentration: 10000 cell/ml
- Number of Replicates: 3

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not required for this kind of test

Test conditions

Hardness:

Not mentioned in the study report; however the test medium was prepared according EEC Directive 92/69/EEC, Annex V, Part C.3. Algal inhibition test updating 29 December 1992 and OECD Guideline for Testing of Chemicals, No. 201 Algal growth inhibition test, June 1984.

Test temperature:

23 °C (max. temperature difference 2 °C)

pH:

The pH value was about pH 8.

Dissolved oxygen:

Not mentioned in the study report; however the test medium was prepared according EEC Directive 92/69/EEC, Annex V, Part C.3. Algal inhibition test updating 29 December 1992 and OECD Guideline for Testing of Chemicals, No. 201 Algal growth inhibition test, June 1984.

Salinity:

Not mentioned in the study report; however the test medium was prepared according EEC Directive 92/69/EEC, Annex V, Part C.3. Algal inhibition test updating 29 December 1992 and OECD Guideline for Testing of Chemicals, No. 201 Algal growth inhibition test, June 1984.

Conductivity:

Not mentioned in the study report; however the test medium was prepared according EEC Directive 92/69/EEC, Annex V, Part C.3. Algal inhibition test updating 29 December 1992 and OECD Guideline for Testing of Chemicals, No. 201 Algal growth inhibition test, June 1984.

Nominal and measured concentrations:

The analytical verifications of the test substance were investigated at different concentrations in OECD - medium. The analytical results yielded 80 % or higher recoveries; they varied between 101 and 108 % of the nominal concentrations at test initiation and between 99 to 113 % at test termination. Therefore, all biological results are related to the nominal concentrations of the test item.

Details on test conditions:

See table below

Reference substance (positive control):

no

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

22.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

52 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 4

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

44.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Key result

Duration:

72 h

Dose descriptor:

EC90

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 7

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass & growth rate

Effect concentrations 8

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: biomass & growth rate

Details on results:

Effect on the development of biomass:
EbC10 (72 h) = 22.2 mg/L
EbC50 (72 h) = 52.0 mg/L
EbC90 (72 h) > 100 mg/L

Effect on growth rate:
ErC10 (72 h) = 44.5 mg/L
ErC50 (72 h) > 100 mg/L
ErC90 (72 h) > 100 mg/L

No observed effect concentration (95% significance level)
NOEC (72 h) = 12.5 mg/L

Lowest observed effect concentration (95% significance level)
LOEC (72 h) = 25.0 mg/L

Results with reference substance (positive control):

• The EbC50 (72 h) of the control substance potassium dichromate was 0.46 mg/L
• The cell multiplication factor in the untreated control was after 72 hours: 90-fold.
• There was no variation of the pH-values within the test period of 72 h about more than 2 units in the control.

The test is valid.

Reported statistics and error estimates:

Determination of the mean fluorescence after 0, 24, 48, 72 h.
Calculation of the growth rate over the total duration of the study for each concentration level and comparison of values of treated samples in relation to untreated samples.
The EC values are calculated (linear regression analysis) from the concentration-response relationship.
The LOEC is determined by comparing the means of the biomass or growth rate of the various concentrations levels with the control. The Duncan multiple range test is carried out at a 95 % significance level. Every higher tested concentration must have at least the same or a stronger effect then the LOEC.
The NOEC is the tested concentration immediately below the LOEC.

Any other information on results incl. tables

Inhibition of the algal biomass and growth rates after 72 h

concentration (mg/L)	biomass		growth rate
	relative	Inhibition (% of the control)	relative	Inhibition (% of the control)
0 (control)	1967	0.0	0.061	0.0
100	497	74.7	0.042	30.6
50	1012	48.5	0.054	11.8
25	1661	15.6	0.060	1.3
12.5	2306	-17.2	0.065	-6.8
6.25	2134	-8.5	0.064	-4.8
3.13	2012	-2.3	0.063	-3.8
1.56	1910	2.9	0.062	-1.3
0.78	1915	2.6	0.062	-1.0
0.39	1900	3.4	0.062	-1.0

Results of concentration control of "Xyligen K 30 F" in "OECD-Medium Teil 9 einfach konzentriert"

Sample no.	Date of sample	Date of sampling	Expected concentration	Value found
01 /0069/60/1/b/h0/K	Jan 29, 2002	Jan 29, 2002	0 mg/L	< 0.1 mg/L
01 /0069/60/1/b/h0/100	Jan 29, 2002	Jan 29, 2002	100 mg/L	101 mg/L
01/0069/60/1/b/h0/6,25	Jan 29, 2002	Jan 29, 2002	6.25 mg/L	6.52 mg/L
01/0069/60/1/b/h0/0,39	Jan 29, 2002	Jan 29, 2002	0.39 mg/L	0.42 mg/L
01 /0069/60/1 /b/h 72/K	Jan 29, 2002	Feb 01, 2002	0 mg/L	< 0.1 mg/L
01/0069/60/1/b/h72/100	Jan 29, 2002	Feb 01, 2002	100 mg/L	100 mg/L
01 /0069/60/1 /b/h 72/6,25	Jan 29, 2002	Feb 01, 2002	6.25 mg/L	6.20 mg/L
01/0069/60/1/b/h72/0,39	Jan 29, 2002	Feb 01, 2002	0.39 mg/L	0.44 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

NOEC: 12.5 mg/L, corresponds to 3.75 mg/L (100% w/w K-HDO)
LOEC: 25 mg/L corresponds to 7.5 mg/L (100% w/w K-HDO)
ErC50: >100 mg/L corresponds to >30 mg/L (100% w/w K-HDO)
EbC50: 52 mg/L corresponds to 15.6 mg/L (100% w/w K-HDO)

Executive summary:

The acute toxicity of N-cyclohexyl-diazeniumdioxy-potassium (respectively Xyligen 30 F (= 30 % K-HDO in water)) to the algae Desmodesmus subspicatus CHODAT SAG 86.81 was determined in a 72-hour static test according to EEC Directive 92/69/EEC, Annex V, Part C.3. Algal inhibition test, updating 29 December 1992 and OECD Guideline for Testing of Chemicals, No. 201 Algal growth inhibition test, June 1984. The following test results were obtained:

Effect on the development of biomass:

EbC10 (72 h) = 22.2 mg/L

EbC50 (72 h) = 52.0 mg/L

EbC90 (72 h) > 100 mg/L

Effect on growth rate:

ErC10 (72 h) = 44.5 mg/L

ErC50 (72 h) > 100 mg/L

ErC90 (72 h) > 100 mg/L

No observed effect concentration (95% significance level)

NOEC (72 h) = 12.5 mg/L

Lowest observed effect concentration (95% significance level)

LOEC (72 h) = 25.0 mg/L

The test substance was tested in the concentration range between 100 and 0.39 mg/L. The dilution factor was 2. The analytical verifications of the test substance were investigated at different concentrations in OECD-medium. The analytical results yielded 80 % or higher recoveries; they varied between 101 and 108 % of the nominal concentrations at test initiation and between 99 to 113 % at test termination. Therefore, all biological results are related to the nominal concentrations of the test item.

VALIDITY CRITERIA

·      The EbC50 (72 h) of the control substance potassium dichromate was 0.46 mg/L

·      The cell multiplication factor in the untreated control was after 72 hours: 90-fold.

·      There was no variation of the pH-values within the test period of 72 h about more than 2 units in the control.

The test is valid.