5-((4-chloro-6-((8-hydroxy-3,6-disulfo-7-((2-sulfo-4-((2(sulfooxy)ethyl)sulfonyl)phenyl)diazenyl)naphthalen-1-yl)amino)-1,3,5-triazin-2-yl)amino)-4-hydroxy-3-((Z)-(3-sulfophenyl)diazenyl)naphthalene-2,7-disulfonic acid

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

experimental data of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: as mentioned below

GLP compliance:

not specified

Analytical monitoring:

not specified

Details on sampling:

WoE 2 and WoE 3:
- Concentrations: 100 mg/l
- Sampling method: Test chemical solution was prepared directly by dissolving the formulation powder in specific test medium for algae (ISO 8692 medium).

Vehicle:

not specified

Test organisms (species):

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

Details on test organisms:

WoE 2 and WoE 3:
TEST ORGANISM
- Common name: green algae
- Method of cultivation: Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium.
- Other: Raphidocelis subcapitata was routinely maintained from a frozen aliquot generated by algae purchased from the national distributor of MicroBioTests Inc.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

WoE 2 and WoE 3: 25 ± 1°C

Nominal and measured concentrations:

WoE 2 and WoE 3: 100 mg/l (Nominal concentrations)

Details on test conditions:

WoE 2 and WoE 3:
TEST SYSTEM
- Test vessel: Microplate was used as a test vessel for the study.
- Material, size, headspace, fill volume: 24-well microplate
- Initial cells density: 10000 cells/ml
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 3 replicates
- No. of vessels per vehicle control (replicates): 3 replicates
- Other: Test vessel was incubated on an orbital shaker (90 rpm) in a thermostatic chamber.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Light intensity and quality: Light was provided by a 2 W LED Unit for each plate.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal cell density was measured at the beginning and end of treatment with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Inc), selecting a gate of 4–14 μm or by visual scoring with a Burker's chamber. Results were expressed as effect on biomass (EbC50) and on growth rate (ErC50).

Reference substance (positive control):

yes

Remarks:

WoE 2 and WoE 3: Potassium dichromate (0.10 to 1.8 mg/l)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WoE 2 and WoE 3

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

135.323 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WoE 2: 95% C. I. – 116.974 to 179.365 mg/l

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

45.365 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: WoE 2

Key result

Duration:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

152.76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: WoE 3: 95% C. I. – 134.406 to 179.274 mg/l

Duration:

72 h

Dose descriptor:

other: EbC50

Effect conc.:

25.904 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: WoE 3: 95% C. I. – 22.233 to 32.632 mg/l

Reported statistics and error estimates:

WoE 2 and WoE 3: All statistical analyses were done using Prism5 (GraphPad Software, Inc.). One-way ANOVA was used for statistical comparisons with Bonferroni's, Dunnett's and Tukey's post hoc tests for multiple comparisons. Significance was set at p<0.05. REGTOX macro Excel™ for dose-response was used to obtain EC50 values by a non-linear regression analysis with Hill's model.

Validity criteria fulfilled:

not specified

Conclusions:

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr ErC50 value can be expected to be in the range 135.323 to 152.76 mg/l, respectively.

Executive summary:

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic algae. The studies are as mentioned below:

Toxicity to aquatic algae study was carried out for 72 hrsfor assessing the effect of test chemical on Raphidocelis subcapitata (green algae). Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Test chemical solution was prepared directly by dissolving the formulation powder in specific test medium for algae (ISO 8692 medium). Limit test was performed using a nominal test chemical conc. of 100 mg/l. Test organism were exposed to the test chemical (2 ml) in sterile 24-well plates for 72 hrs under static conditions. Algal stock solution containing 1010000 cells/mL was added (20 μL) to each well to obtain a starting algal density of 10000 cells/mL. Untreated ISO formulation freshwater and Potassium dichromate (0.10 to 1.8 mg/l) were used as a negative and positive control. All experiments was performed in triplicates. Plates were incubated at 25±1 °C for 72 hr on an orbital shaker (90 rpm) in a thermostatic chamber. Light was provided by a 2 W LED Unit for each plate. Algal cell density was measured at the beginning and end of treatment with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Inc), selecting a gate of 4–14 μm or by visual scoring with a Burker's chamber. Results were expressed as effect on biomass (EbC50) and on growth rate (ErC50). All statistical analyses were done using Prism5 (GraphPad Software, Inc.). One-way ANOVA was used for statistical comparisons with Bonferroni's, Dunnett's and Tukey's post hoc tests for multiple comparisons. Significance was set at p<0.05. REGTOX macro Excel™ for dose-response was used to obtain EC50 values by a non-linear regression analysis with Hill's model. On basis of effect of the test chemical on growth rate of the test organism Raphidocelis subcapitata, the 72 hr ErC50 value was determined to be 135.323 mg/l (95% C. I. – 116.974 to 179.365 mg/l) & on the basis of biomass, the 72 hr EbC50 value was determined to be 45.365 mg/l, respectively.Thus, based on the ErC50 value (135.323mg/l) alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

Another toxicity to aquatic algae study was carried out for 72 hrsfor assessing the effect of test chemical on Raphidocelis subcapitata (green algae). Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Test chemical solution was prepared directly by dissolving the formulation powder in specific test medium for algae (ISO 8692 medium). Limit test was performed using a nominal test chemical conc. of 100 mg/l. Test was conducted using the same test procedure and test conditions as mentioned in above study. On basis of effect of the test chemical on growth rate of the test organism Raphidocelis subcapitata, the 72 hr ErC50 value was determined to be 152.76 mg/l (95% C. I. – 134.406 to 179.274 mg/l) & on the basis of biomass, the 72 hr EbC50 value was determined to be 25.904 mg/l (95% C. I. – 22.233 to 32.632 mg/l), respectively. Thus, based on the ErC50 value (152.76 mg/l) alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr ErC50 value can be expected to be in the range 135.323 to 152.76 mg/l, respectively. Thus, based on the ErC50 value alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

Description of key information

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr ErC50 value can be expected to be in the range 135.323 to 152.76 mg/l, respectively

Key value for chemical safety assessment

EC50 for freshwater algae:

135.32 mg/L

Additional information

Data available of the structurally and functionally similar read across chemicals has been reviewed to determine the effect of the test chemical on aquatic algae. The studies are as mentioned below:

 

Toxicity to aquatic algae study was carried out for 72 hrs for assessing the effect of test chemical on Raphidocelis subcapitata (green algae). Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Test chemical solution was prepared directly by dissolving the formulation powder in specific test medium for algae (ISO 8692 medium). Limit test was performed using a nominal test chemical conc. of 100 mg/l. Test organism were exposed to the test chemical (2 ml) in sterile 24-well plates for 72 hrs under static conditions. Algal stock solution containing 1010000 cells/mL was added (20 μL) to each well to obtain a starting algal density of 10000 cells/mL. Untreated ISO formulation freshwater and Potassium dichromate (0.10 to 1.8 mg/l) were used as a negative and positive control. All experiments was performed in triplicates. Plates were incubated at 25±1 °C for 72 hr on an orbital shaker (90 rpm) in a thermostatic chamber. Light was provided by a 2 W LED Unit for each plate. Algal cell density was measured at the beginning and end of treatment with a TC20™ Automated Cell Counter (Bio-Rad Laboratories, Inc), selecting a gate of 4–14 μm or by visual scoring with a Burker's chamber. Results were expressed as effect on biomass (EbC50) and on growth rate (ErC50). All statistical analyses were done using Prism5 (GraphPad Software, Inc.). One-way ANOVA was used for statistical comparisons with Bonferroni's, Dunnett's and Tukey's post hoc tests for multiple comparisons. Significance was set at p<0.05. REGTOX macro Excel™ for dose-response was used to obtain EC50 values by a non-linear regression analysis with Hill's model. On basis of effect of the test chemical on growth rate of the test organism Raphidocelis subcapitata, the 72 hr ErC50 value was determined to be 135.323 mg/l (95% C. I. – 116.974 to 179.365 mg/l) & on the basis of biomass, the 72 hr EbC50 value was determined to be 45.365 mg/l, respectively. Thus, based on the ErC50 value (135.323mg/l) alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

Another toxicity to aquatic algae study was carried out for 72 hrs for assessing the effect of test chemical on Raphidocelis subcapitata (green algae). Algae were routinely cultured at 25±1 °C with light in Cyanobacteria BG-11 Freshwater Solution. The culture was split twice a week, adding fresh medium. Test chemical solution was prepared directly by dissolving the formulation powder in specific test medium for algae (ISO 8692 medium). Limit test was performed using a nominal test chemical conc. of 100 mg/l. Test was conducted using the same test procedure and test conditions as mentioned in above study. On basis of effect of the test chemical on growth rate of the test organism Raphidocelis subcapitata, the 72 hr ErC50 value was determined to be 152.76 mg/l (95% C. I. – 134.406 to 179.274 mg/l) & on the basis of biomass, the 72 hr EbC50 value was determined to be 25.904 mg/l (95% C. I. – 22.233 to 32.632 mg/l), respectively. Thus, based on the ErC50 value (152.76 mg/l) alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.

 

On the basis of the experimental studies of the structurally and functionally similar read across chemical and applying the weight of evidence approach and by evaluating the effect of test chemical on test organism, the 48 hr ErC50 value can be expected to be in the range 135.323 to 152.76 mg/l, respectively. Thus, based on the ErC50 value alongwith growth rate effect, test chemical was considered as non-toxic to aq. algae and hence, considered to be ‘not classified’ as per CLP classification criteria.