Disodium 2,2'-[(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[3-bromo-5-butyltoluene-4-sulphonate]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2018 to 31 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Frequency of measurement: At the start of exposure, 24 and 48 hours after the start of exposure, and the end of exposure.
- Sample for measurement : Another solution was sampled separately from the preparation container at the start of exposure. The test solution was taken out from the test vessel in each test level for analytical chemistry at 24 and 48 hours after the start of exposure. The mixed solution was taken out with equal volume of the test solution from the test vessels in each test level at the end of exposure.
- Removal of algae: Centrifugation (3000 rpm, 10 minutes) (24 and 48 hours after the start of exposure, and the end of exposure)
- Volume of sample: Approximately 10 mL from all test levels at the start of exposure, 3 mL from all test levels at 24 and 48 hours after the start of exposure and 9 mL from all test levels, at the end of exposure.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- The weighed test sample of 70.0 mg and medium of 630 mL were mixed to produce the nominal concentration of 100 mg/L and stirred by a magnetic stirrer. Then, it was filtered with a glass fibre filter (GB-140, 0.4 µm pore size, Toyo Roshi) by suction. The filtrate was used as the stock solution. The test solution was prepared in a preparation container by mixing the required volume of the stock solution and the medium processing similar to the control and divided into each test vessel. In stock solution content of 100 % level, the stock solution was divided into test vessel.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: ATCC 22662
- Source: American Type Culture Collection
- Method of cultivation: Passage cultured under sterile conditions in this test facility

ACCLIMATION
- The algae were counted in pre-culture incubated under the same conditions as the test for 3 days (from August 25 to August 28, 2018) as inoculums.
- The deformed and/or abnormal cells in the pre-culture used for the study were not present in the test flasks.
- Culturing media and conditions: same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.2 - 22.5 °C

pH:

7.8 - 7.9

Nominal and measured concentrations:

Nominal: 100, 31.6, 10.0, 3.16, 1.00, 0.316 and 0.100 % stock solutions
Geometric mean measured: 93.6, 27.4, 7.27, 2.09, 0.469, 0.0939 and 0.00163 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterilised 500 mL Erlenmeyer flask (with gas-permeable Silicosen®)
- Flasks were incubated with rotary shaking (approximately 100 rpm)
- Fill volume: 50 mL/ vessel
- Initial cells density: 0.75 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- One additional replicate for analytical chemistry of the test material was set

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: At the pre-culture and algae growth inhibition study, the OECD medium (OECD 201) was prepared with purified water and included the following components: H3BO3 0.185 mg/L, MnCl2.4H2O 0.415 mg/L, ZnCl2 0.00300 mg/L, FeCl3.6H2O 0.0640 mg/L, Na2EDTA.2H2O 0.100 mg/L, CoCl2.6H2O 0.00150 mg/L, Na2MoO4.2H2O 0.00700 mg/L, CuCl2.2H2O 0.00001 mg/L, CaCl2.2H2O 18.0 mg/L, NH4Cl 15.0 mg/L, KH2PO4 1.60 mg/L, NaHCO3 50.0 mg/L, MgCl2.6H2O 12.0 mg/L and MgSO4.7H2O 15.0 mg/L.
- Intervals of water quality measurement: The appearance of the test solution was observed at the start and the end of exposure. pH was measured from the preparation container at the start of exposure and in one test vessel in each test level at the end of exposure. Culture temperature was measured at the start, 1-day and 2-days after the start, and at the end of exposure. Light intensity was measured at the start, 1-day and 2-days after the start, and at the end of exposure in the incubator.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous illumination provided by alternative LED light to fluorescent lamp
- Light intensity and quality: Nominal 90 µmol.m^-2.s^-1 in wavelength range of 400-700 nm within ± 20 % of nominal (within ± 15 % from the average light intensity)

EFFECT PARAMETERS MEASURED:
- Biomass (cell concentration) was measured every 24 hours after the start of exposure using a particle counter (Model COULTER Z2).
- Observation of cell condition was recorded in one vessel in each test level at the end of exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: a geometric series with a factor of √10
- The test concentrations and the factor were decided based on the results of preliminary study.
- The preliminary study was performed at a nominal concentration of 100 mg/L. From the preliminary test results, it was suggested that the effect might differ depending on the volume of test solution at 100 % level as stock solution content. Therefore, the study using 50 mL of test solution volume, which reduces the light path is conducted in definitive study. Also, since it is expected that the concentration of the test material in the test solution decrease during the exposure, the concentration of the test material in test solution is measured at the start, 24-hour after and 48-hour after and the end of exposure.

TREATMENT OF THE RESULTS
- The results of study were estimated by geometric mean of the measured concentration of test material in the test solution during the exposure.
- Calculation of concentration-inhibition rates:
The mean value of biomass for each test level was plotted against time to produce growth curves. Using this curve, inhibition rates were calculated comparing with control values on growth rate.
Comparison of growth rates: The specific growth rate for a specific period was calculated as the logarithmic increase in biomass according to the following formula:
µi-j = (lnXj – lnXi) / (tj – ti)
Where:
µi-j = Specific growth rate from time i to j (normally d^-1)
Xi = Value of biomass at ti: Set value of biomass was used at the start of exposure (t0).
Xj = Value of biomass at tj
ti = Time (d) of ith measurement after beginning of exposure
tj = Time (d) of jth measurement after beginning of exposure
Specific growth rate over the exposure duration (0-72h) was calculated for determination of EC50 and NOEC. In the control, specific growth rates for section-by-section were calculated for check of validity of the test.
The percentage inhibition for each exposure level was mean value of the percent inhibition in average specific growth rate for a replicate (Iµ) in test level. The percent inhibition (Iµ) was calculated from mean value for average specific growth rate in control (µc), average specific growth rate for each replicate in exposure level (µr), and the following formula:
Iµ = [(µc - µT) / µc] x100

- Calculating method of EC50:
The EC50 was estimated as "> the highest test concentration" since no less than 50 % of inhibition rate was not obtained within the exposure levels. The EC50 was denoted as ErC50 based on growth rate.
The EC50 was determined using computer program (running on Microsoft software "Excel") constructed by the test facility.

- Estimation of NOEC:
Regarding the growth rate, Bartlett's test was done to determine the homogeneity of variance for the data. Then one-way analysis of variance and Dunnett's multiple comparison test was used to determine the significant difference between the control and the exposure levels. The statistical analysis was conducted using computer program (running on Microsoft software "Excel'') constructed by our test facility. The NOEC was determined by the results of statistical analysis and cell condition.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 96.3 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.469 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

OBSERVATIONS AND MEASUREMENTS OF THE TEST SOLUTION
- Appearance of test solution: At the start of exposure, test solutions of 0.0939 and 0.00163 mg/L levels were colourless and clear. The appearance of test solution in the other exposure levels were clear blue depending on the concentration. At the end of exposure, the appearance of test solution in 93.6, 27.4 and 7.27 mg/L levels were blue depending on the concentration, that in 2.09 mg/L level was a little greenish light blue, that in 0.469 mg/L level was a little bluish green and those in the other exposure levels were green, due to the algal growth. The solution of control was colourless and clear at the start of exposure, and it was green at the end of exposure due to the algal growth.
- Water quality and environmental conditions: The measured values of pH were 7.8-7.9. Culture temperatures in the incubator were 22.2-22.5 °C and light intensities were 88-90 µmol.m^-2·s^-1.
- Concentration of test material in test solution: The measured concentrations of test material in the test solutions were 0.0761-102 mg/L at the start of exposure, less than determination limit (< 0.0180 mg/L)-92.0 mg/L at 24 hours after the start of exposure, less than determination limit (< 0.0180 mg/L)-91.6 mg/L at 48 hours after the start of exposure, and less than determination limit (< 0.0180 mg/L)-93.0 mg/L at the end of exposure. In 0.316-100 % levels as stock solution content, which were quantifiable during the exposure, those were 24.9-90.1 %, 26.9-89. 7 % and 27.3-91.1 % at the start of exposure, respectively.

ErC50
- ErC50 of the test material based on the growth rate was > 93.6 mg/L.

GROWTH CURVE IN EACH TEST LEVEL, CELL OBSERVATIONS AND NOEC
- The algal growth in 93.6, 27.4 and 7.27 mg/L levels were logarithmic, although growth inhibition was found. In 2.09 mg/L level, algae grew similar to the control although a little growth inhibition was found. The algal growth in 0.469-0.00163 mg/L levels were same as the control.
- The following results of cell observation were based on the comparison with the control. Some bloated cells were observed in 93.6 and 27.4 mg/L levels. The conditions of cells in the other exposure levels were same as the control. In the control, the condition of cells was not abnormal.
- On the growth rate, there were statistical differences in 93.6-2.09 mg/L levels. By the results in statistical analysis and cell observation showed above, NOEC based on the growth rate was estimated at 0.469 mg/L.

VALIDITY OF THE TEST
- Growth of control: The cells in the control grew exponentially during the exposure. At the end of exposure, it increased to a factor of 96 or more of the number of initial cells in the control. This meets the validity of test: the cell growth in the control should have increased by a factor of at least 16 at 72 hours after the start of exposure.
- Specific growth rates of section-by-section in controls: The mean coefficient of variation for section-by-section specific growth rates in the control cultures was 11 %. It meets the validity of test: the mean coefficient of variation in the control cultures must not exceed 35 %.
- Specific growth rates in replicate controls: The coefficient of variation of specific growth rates in replicate control cultures was 0.61 %. It meets the validity of test: the coefficient of variation in control cultures must not exceed 7 %.

Results with reference substance (positive control):

An algae growth inhibition study of a reference substance with the test organisms was periodically conducted.
ErC50 (0-3d): 1.3 mg/L
This value was within the stipulated range [mean ± 2 S.D.: 0.61-1.4 mg/L (n=32, fluorescent lights)] to background data in this test facility.

Table 1: Growth Rate and Growth Inhibition Rate

Measured Concentration (mg/L)	No.	Growth Rate (0-3 d)	Growth Inhibition Rate (%)
Control	A	1.54	-
	B	1.55	-
	C	1.54	-
	D	1.55	-
	E	1.54	-
	F	1.52	-
	Mean	1.54	-
	S.D.	0.00940	-
0.00163	A	1.54	-0.32
	B	1.53	0.27
	C	1.56	-1.1
	Mean	1.54	-0.38
	S.D.	0.0106	0.69
0.0939	A	1.54	0.065
	B	1.55	-0.61
	C	1.54	-0.26
	Mean	1.54	-0.27
	S.D.	0.00518	0.34
0.469	A	1.53	0.49
	B	1.53	0.52
	C	1.55	-0.45
	Mean	1.54	0.19
	S.D.	0.00842	0.55
2.09	A	1.49	3.3
	B	1.45	5.4
	C	1.50	2.2
	Mean	1.48	3.7
	S.D.	0.0253	1.6
7.27	A	1.17	24
	B	1.16	25
	C	1.14	26
	Mean	1.16	25
	S.D.	0.0159	1.0
27.4	A	1.04	32
	B	1.06	31
	C	1.03	33
	Mean	1.04	32
	S.D.	0.0159	1.0
93.6	A	0.792	48
	B	0.833	46
	C	0.829	46
	Mean	0.818	47
	S.D.	0.0223	1.5

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the ErC50 was > 93.6 mg/L and NOEC was 0.469 mg/L.

Executive summary:

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guideline OECD 201, under GLP conditions.

Pseudokirchneriella subcapitata were exposed to the test material at seven test concentrations of 100, 31.6, 10.0, 3.16, 1.00, 0.316 and 0.100 % as stock solution content (a geometric series with a factor of√10 (93.6, 27.4, 7.27, 2.09, 0.469, 0.0939 and 0.00163 mg/L as measured concentration, respectively), and a control for 72 hours.

Since the test material was an ultramarine powder and test solutions in exposure levels were blue and clear in a concentration-dependent manner, effect on growth caused by colouring was confirmed in preliminary study. As the result, it was considered that there was slight effect caused by colouring. The definitive study was carried out by 50 mL test solution (half of usual volume). On observation of cell condition at the end of exposure, some bloated cells were observed in 93.6 and 27.4 mg/L levels. In these levels, the mean growth inhibition rates were 47 and 32 %, respectively. Therefore, it is considered that the test material affected algal cell division in these exposure levels. The test material concentration in test solution was reduced during the exposure, and that at the end of exposure was 27.3-91.1 % of the concentration at the start (in 100-0.316 % as stock solution content). So, the results of study were estimated by geometric mean of the measured concentrations of test material in test solution during the exposure.

The environmental conditions were within the suitable range. Therefore, it is concluded that this study complied with the applied test guidelines.

Under the conditions of this study, the ErC50 was > 93.6 mg/L and NOEC was 0.469 mg/L.

Description of key information

Under the conditions of the study, the ErC50 was > 93.6 mg/L and NOEC was 0.469 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

0.469 mg/L

Additional information

The toxicity of the test material to aquatic algae was investigated in accordance with the standardised guideline OECD 201, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Pseudokirchneriella subcapitata were exposed to the test material at seven test concentrations of 100, 31.6, 10.0, 3.16, 1.00, 0.316 and 0.100 % as stock solution content (a geometric series with a factor of√10 (93.6, 27.4, 7.27, 2.09, 0.469, 0.0939 and 0.00163 mg/L as measured concentration, respectively), and a control for 72 hours.

Since the test material was an ultramarine powder and test solutions in exposure levels were blue and clear in a concentration-dependent manner, effect on growth caused by colouring was confirmed in preliminary study. As the result, it was considered that there was slight effect caused by colouring. The definitive study was carried out by 50 mL test solution (half of usual volume). On observation of cell condition at the end of exposure, some bloated cells were observed in 93.6 and 27.4 mg/L levels. In these levels, the mean growth inhibition rates were 47 and 32 %, respectively. Therefore, it is considered that the test material affected algal cell division in these exposure levels. The test material concentration in test solution was reduced during the exposure, and that at the end of exposure was 27.3-91.1 % of the concentration at the start (in 100-0.316 % as stock solution content). So, the results of study were estimated by geometric mean of the measured concentrations of test material in test solution during the exposure.

The environmental conditions were within the suitable range. Therefore, it is concluded that this study complied with the applied test guidelines.

Under the conditions of the study, the ErC50 was > 93.6 mg/L and NOEC was 0.469 mg/L.