Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)

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

Study period:

08 December 2017 - 22 January 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

23 March 2006

Deviations:

yes

Remarks:

methodological limitations, e.g. discrepancy between results of solubility test and actually chosen test conc., inconsistent analytical procedures

Principles of method if other than guideline:

- The test item was not tested at a concentration above 5 mg/L.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)

EC Number:

250-774-0

EC Name:

Hydrogen bis[1-[(5-chloro-2-hydroxyphenyl)azo]-2-naphtholato(2-)]chromate(1-)

Cas Number:

31714-55-3

Molecular formula:

C32H19Cl2CrN4O4

IUPAC Name:

[1-{[5-chloro-2-(hydroxy-kappaO)phenyl]diazenyl}-2-naphtholato(2-)-kappaO]{1-[(5-chloro-2-hydroxyphenyl)diazenyl]-2-naphtholato-kappaO}chromium

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Black powder
- Storage conditions: At room temperature

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: test samples from each replicate were drawn and pooled and mixed together for each control and concentration
- Volume: 10 mL (from each replicate)
- Frequency: at 0 and 72 h
- Sample storage conditions before analysis: not applicable, test samples were analysed directly

Test solutions

Vehicle:

yes

Remarks:

dimethyl formamide

Details on test solutions:

TEST FOR SOLUBILITY:
Before conducting the preliminary range finding study, the test item was checked for solubility and precipitation in algal medium, acetone and dimethyl formamide (DMF). Based on the results, DMF was chosen as the appropriate vehicle for the test item.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct application to the medium: a quantity of 60.0 and 24.0 mg test item was dissolved in 1 mL DMF to achieve test concentrations of 60.0 mg/mL (Stock A) and 24.0 mg/mL (Stock B). A quantity of 96.0, 38.4 and 15.6 mg test item was dissolved in a small volume of DMF, transferred into a 10 mL volumetric flask and the volume was made up to 10 mL with DMF to achieve test concentrations of 9.6 mg/mL (Stock C), 3.84 mg/mL (Stock D) and 1.56 mg/mL (Stock E).
Volumes of 10 µL from the stock solutions A to E and 1500 µL algal culture were taken and diluted to 120 mL with sterile algal culture medium in respective flasks to obtain the nominal test concentrations of 0.13, 0.3, 0.8, 2.0 and 5.0 mg test item/L.
- Controls: For the control group, only 1500 µL alga culture was transferred using a micropipette and diluted to 120 mL with sterile algal culture medium and for the vehicle control group, 10 µL dimethyl formamide and 1500 µL algal culture were taken and diluted to 120 mL with sterile algal culture medium.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection, 10801, University of Boulevard, Manassas, Virginia, 20110-2209, USA
- Age of inoculum (at test initiation): not specified
- Method of cultivation: static condition

ACCLIMATION
- Acclimation period: 2 days
- Culturing media and conditions: same as the test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

21 - 23 °C

pH:

7.09 - 7.41

Nominal and measured concentrations:

Nominal concentrations: 0.13, 0.32, 0.8, 2.0 and 5.0 mg/L
Measured concentrations: 0.15, 0.33, 0.83, 2.06, 5.01 mg/L at t=0 and 0.11, 0.31, 0.82, 1.91, 4.75 mg/L at t=72, respectively, corresponding to above nominal concentrations.
Measured concentrations at t=72 h were 82-104% of nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL sterile conical flask
- Type (delete if not applicable): open
- Aeration: no
- Initial cells density: 6313
- Control end cells density: 821667 (blank control) and 849167 (vehicle control)
- No. of vessels per concentration: 3 replicates
- No. of vessels per control: 6 replicates
- No. of vessels per vehicle control: 6 replicates

GROWTH MEDIUM
- Standard medium used: yes, according to OECD 201

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: sterilized deionised water was used to prepare the medium
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: not applicable
- Light intensity and quality: 6500-6710 lux

EFFECT PARAMETERS MEASURED: yield and growth rate at 0, 24, 48 and 72 h
- Determination of cell concentrations: manual counting using a haemocytometer and a microscope
The no. of cells in 1 mL was calculated as follows:
no. of cells = (total no. of cells counted*dilution factor*1000)/(area counted*depth of fluid)
Where dilution factor = 1.0; area counted = 4 x 1 mm^2 (constant); depth of fluid = 0.1 mm (constant)
- Other: pH of the algal test media was measured at test start (0 h) and test end (72 h). Temperature and illumination were recorded daily

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study
- Test concentrations: 0.0, 0.001, 0.01, 0.1, 1.0 and 5.0 mg/L
- Results used to determine the conditions for the definitive study: Yes, the inhibition of growth rate was 0.59, 0.73, 0.88, 9.54 and 20.12% at the test concentrations of 0.001, 0.01, 0.1, 1.0 and 5.0 mg/L, respectively. Based on these results, the EC50 for growth rate was expected to exceed the highest tested concentration of 5.0 mg/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (January 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.89-2.17 mg/L

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Results showed that at the start and the end of the test, the test item was in the range of 82.3 -113.6% of nominal concentrations (72.4 -99.1% of initial measured concentrations at test end). Therefore, effect parameters were based on nominal concentrations.
- At the highest tested concentration (5.0 mg/L) a growth rate inhibition of 20.7% was observed. Therefore, the ErC50 was determined to exceed the highest test concentration.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Tested concentrations: 0.0, 0.1, 0.3, 0.8, 2.0 and 5.0 mg/L
- 72h-ErC50: 1.29 mg/L (95% confidence limits 1.12-1.49 mg/L)

Reported statistics and error estimates:

NOEC and LOEC were determined by a suitable statistical procedure for multi-sample comparison. Data of biomass, specific growth rate and yield were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett’s Test through in-house developed, validated computer software.
The EC10, EC20, and EC50 were calculated using the Probit analysis method (Finney, 1971) using in-house developed, validated software

Any other information on results incl. tables

Table 1 Measured concentrations of T-95/T-99 in Algal Medium (main study)

Groups	Nominal Conc. based on Purity of Test Item (mg/L)	t=0 h			t=72 h
		Peak Area of Sample (Y)	Analysed Conc.(mg/L)	Recovery^# (%)	Peak Area of Sample (Y)	Analysed Conc.(mg/L)	Recovery^# (%)
G1 (0.00)	0.00000	ND	0.00000	0.00	ND	0.00000	0.00
G2 (0.00)	0.00000	ND	0.00000	0.00	ND	0.00000	0.00
G3 (0.13)	0.12922	11813	0.14686	113.65	10249	0.10629	82.26
G4 (0.32)	0.31808	27221	0.33284	104.64	26772	0.30986	97.42
G5 (0.80)	0.79520	68448	0.83044	104.43	68430	0.82311	103.51
G6 (2.00)	1.98800	170443	2.06151	103.70	156260	1.90521	95.84
G7 (5.00)	4.97000	82706	5.01266	100.86	78673	4.74652	95.50
Intercept of Y-axis (a)		-354.73			1621.99
Slope of the line (b)		82850.90			81165.80

ND = Not Detected, Conc. = Concentration.

^#relative to nominal

Dilution factor for the sample of groups G1, G2, G3, G4, G5, G6 and G7 were 1.0, 1.0, 1.0, 1.0, 1.0, 1.0 and 5.0, respectively.

Table 2 Percentage (%) inhibition of biomass, growth rate and yield (main study)

Group	Nominal Test Concentration (mg/L)	Percentage (%) Inhibition of
		Biomass (EbC) 0 - 72 h	Growth Rate (ErC) 0 - 72 h	Yield (EyC)0 - 72 h
G1	0.0 (Control)	-	-	-
G2	0.0 (vehicle control)	-	-	-
G3	0.13	3.02	0.73	3.66
G4	0.32	15.29	3.52	15.82
G5	0.8	34.84	9.25	36.48
G6	2.0	41.08	12.04	44.49
G7	5.0	60.42	20.70	64.07

- = Not Applicable, h = Hour
Note:   Growth rate is a logarithmic term. Therefore, E_bC and E_rC values are not numerically comparable. All values mentioned above are based on the nominal concentrations selected for the study.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'overall remarks' section

Conclusions:

The 72h-NOErC and the 72h-ErC50 of T-95/T-99 were determined to be 0.32 mg/L and >5.0 mg/L, respectively, based on nominal concentrations.

Executive summary:

In an algal toxicity test performed according to OECD 201 and GLP principles, T-95/T-99 was assessed for its effect on the growth of Pseudokirchneriella subcapitata.

The test item was initially tested for solubility and dimethyl formamide was chosen as the most appropriate vehicle for the test item. Nominal concentrations of 0.13, 0.32, 0.8, 2.0 and 5.0 mg/Lwere chosen for the main test and a blank control as well as a vehicle control were included. The test period was 72 hours, under static conditions, in which algal cultures were counted at t=24, t=48 and t=72 h. Test samples for analysis of test concentrations were taken at t=0 and t=72 h. Results showed that test item concentrations were in the range of 82.3 -113.6% of nominal concentrations, and 72.4 -99.1% of initial measured concentrations at test end. Based on these results, effect parameters were based on nominal concentrations.

The 72h-NOErC and the 72h-ErC50 of T-95/T-99 towards Pseudokirchneriella subcapitata were determined to be 0.32 mg/L and >5.0 mg/L, respectively.