Disodium 4,4'-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1'-biphenyl]-2,2'-disulphonate

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:

03 June 2016 - 05 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2008 (ammended by EC No. 2016/266)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23.

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of certificate: 3 Nov 2015

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: yellow/brown powder
- Storage conditions: in refrigerator (2-8°C)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all test concentrations and the control according to the schedule below.
Frequency: at t=0, t=24 and t=72
Volume: 3.0 mL
Storage: Samples were stored in a freezer (≤ 15°C) until analysis.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the Quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: All test concentrations were prepared separately and started with loading rates ranging from 1.0 to 100 mg/L applying three days of magnetic stirring to reach maximum dissolution of the test item in the test medium. The resulting aqueous mixtures were filtered through 0.45 µm membrane filters (Whatman;RC55) where after the Water Accommodated Fractions (WAFs) were used for testing. The final test solutions were clear and increasingly yellow with increasing loading rate.
After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

- Controls: test medium without test item or other additives (blank-control)

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture

CULTIVATION:
- Method: algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Light intensity: 60 to 120 µE/m^2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm
- Medium different from test medium: yes, M1

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Acclimation medium different from test medium: no, M2 (according to OECD 201)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3 mg/L

Test temperature:

22 - 23°C

pH:

At test start: 8.2
At test end: 8.2 - 8.3

Nominal and measured concentrations:

Nominal test concentrations:WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L
Measured test concentrations (TWA): 0.46, 1.8, 6.5, 6.4, 15 and 51 mg/L for WAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/L respectively.

The concentrations remained stable during the first 24 hours of exposure (81-90% of initial) and fairly stable during the remainder of the test (79-89% of initial at the end of the 72-hour exposure period). Concentrations taken from the WAF at 10 mg/L with and without algae were similar during the test period. For more details, see table 1 in section 'Any other information on materials and methods'.
Based on these results, a worst case scenario was followed and the time weighted average (TWA) exposure concentrations were calculated and used for the determination of the effect parameters.

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, open, fill volume: 50 mL
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 138.6 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- Other: 1 extra replicate of each test concentration and the control for sampling purposes; 1 or 2 replicates of each test concentration without algae.

TEST MEDIUM / WATER PARAMETERS
- Standard test medium used: yes, M2
- Source of dilution water: Milli-RO water
- Culture medium different from test medium: yes, M1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: continuous illumination using TLD-lamps with a light intensity within the range of 82 to 87 µE/m^2/s

EFFECT PARAMETERS MEASURED: growth rate at 72 hours.
- Additional measurements: pH at the beginning and at the end of the test. Temperature: continuously in a temperature control vessel; Appearance of the cells: at the end of the final test in the highest concentration by microscopic observations.
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank. One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval.

COMBINED LIMIT/ RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (July 2016)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 51 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

51 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Details on results:

- No ErC50-values could be calculated because the test item proved to be non-toxic (EC50> maximum soluble concentration tested).
- Exponential growth in the control (for algal test): yes
- Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the highest concentration when compared to the control.

- Recovery: measured concentrations ranged from 80 - 98 % of initial loading rates at t=24 and 79 - 89% of initial loading rates at t=72

Individual pH and temperature values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- ErC50: 1.0 mg/L (95%-CI: 0.97-1.0 mg/L)
- EyC50: 0.43 mg/L (95%-CI: 0.42 - 0.44 mg/L)
- Other: results fell within the historical range.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate (Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm, α=0.05, one-sided, smaller) or yield (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller).

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Any other information on results incl. tables

Table 1 Loading rates, measured and TWA concentrations

WAF at x mg/L	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
1.0	0.550	0.448	0.442	0.46
3.2	2.02	1.79	1.77	1.8
10	7.13	6.41	6.35	6.5
10*	6.77	6.62	6.00	6.4
32	18.1	14.5	14.2	15
100	57.9	50.4	48.0	51

*Without algae

Table 2 Percentage inhibition of growth rate (total test period) during the final test

TWA concentration	Mean	Std. Dev.	N	% inhibition
Control	1.644	0.0129	6
0.46	1.645	0.0569	3	-0.1
1.8	1.608	0.0236	3	2.2
6.5	1.565	0.0253	3	4.8
15	1.584	0.0081	3	3.6*
51	1.546	0.0095	3	5.9*

* Effect was statistically significant but biologically insignificant (<10%)

Table 3 Percentage inhibition of growth rate at different time intervals during the final test

TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.557		1.722		1.652
0.46	3	1.798	-15.5	1.543	10.4	1.595	3.4
1.8	3	1.833	-17.7	1.345	21.9	1.645	0.4
6.5	3	1.890	-21.4	1.208	29.9	1.596	3.3
15	3	1.691	-8.7	1.496	13.2	1.565	5.2
51	3	1.830	-17.6	1.214	29.5	1.594	3.5

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall Remarks' section for more details on validity criteria.

Conclusions:

Due to low solubility of the test item, concentration levels that might be toxic for algae could not be reached. The 72h-EC50 for inhibition of both growth rate (ErC50) was >51 mg/L. The 72h-NOEC for growth rate inhibition was 51 mg/L, based on biological significance (<10% effect).

Executive summary:

A study was performed to assess the effect of Disodium 4,4’-bis[[1-[[(2,4-dimethylphenyl)amino]carbonyl]-2-oxopropyl]azo][1,1’-biphenyl]-2,2’-disulphonate on the growth rate of fresh water algae ( Pseudokirchneriella subcapitata ) after 72 hours of exposure. The study was conducted in accordance with OECD 201 and GLP.

 

All test concentrations were prepared separately at loading rates ranging from 1.0 to 100 mg/L by applying three days of magnetic stirring to reach maximum dissolution of the test item in the test medium. The resulting aqueous mixtures were filtered through 0.45 µm membrane filters where after the Water Accommodated Fractions (WAFs) were used for testing.

 

In the final test, six replicates of exponentially growing algal cultures were exposed to an untreated control and three replicates per group were exposed to WAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/L. The initial algal cell density was 10^4cells/mL. The total exposure period was 72 hours. Samples were taken and analysed for confirmation of actual exposure concentrations at the start, after 24 and 72 hours of exposure. Test concentrations remained stable during the first 24 hours of exposure (81-90% of initial) and fairly stable during the remainder of the test (79-89% of initial at the end of the 72-hour exposure period). Based on these results, a worst case scenario was followed and the time weighted average (TWA) exposure concentrations were calculated to be 0.46, 1.8, 6.5, 15 and 51 mg/L. These TWA concentrations were used for the determination of the effect parameters.

 

Due to low solubility of the test item, concentration levels that might be toxic for algae could not be reached. The 72h-EC50 for inhibition of growth rate (ErC50) was >51 mg/L. The 72h-NOEC for growth rate inhibition was 51 mg/L, based on biological significance ( <10% effect).

All acceptability criteria were met and therefore the study was considered to be valid.