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Toxicity to aquatic algae and cyanobacteria

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Reference
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
Justification for type of information:
Experimental test result performed according to the guideline.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
This study was designed to access the toxic effects of the test compound 2,3-dimethylphenol (526-75-0) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
The test solution was prepared in aseptic condition. The test item 2,3-xylenol was prepared by adding 4 mg of test item in 250 ml of BBM to get the final concentration of 16 mg/L. This stock
solution was kept for stirring for 05 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit.The remaining test solutions were prepared by dilution from the above stock solution. To have a better growth and visibility of cells, the initial cell density of the culture was kept 1 X 104cells/ml.Care was taken to have a homogeneous solution for the experiment.
Test organisms (species):
Chlorella vulgaris
Details on test organisms:
TEST ORGANISM
- Common name: green alga
- Source (laboratory, culture collection): National Environmental Engineering Research Institute (NEERI), Nagpur (Laboratory)
- Method of cultivation: Bold’s Basal Medium(BBM)


ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
24, 48, 72 hrs
Test temperature:
22 °C±2°C
Nominal and measured concentrations:
0.5mg/l,1mg/l,2mg/l,4mg/l,8mg/l,16mg/l. All the six concentration were in geometric series spaced by a factor of 2.
Details on test conditions:
TEST SYSTEM
- Test vessel: Conical flasks
- Material, size, headspace, fill volume: 100 ml conical flasks filled with 60 ml was used for the study.
- Initial cells density: 10000cells/ml
- No. of organisms per vessel: 10000cells/ml
- No. of vessels per concentration (replicates): Two replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: The medium to be used for the growth of algae was Bold’s Basal Medium (BBM). It is a medium composed of macronutrients, micronutrients, alkaline EDTA solution and Iron solution. Stock solution of each of these was prepared separately and then a complete medium was prepared and sterilized. De-ionized water was used to prepare the BBM.


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: Yes
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Spectrophotometer - The absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.
- Chlorophyll measurement: No data
- Other: The cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to
observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Test concentrations: Six test concentration were: 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l,100mg/l and 200mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: Mortality of test organisms


Other:
Incubation :
1. The temperature of the orbital shaking incubator was kept constant throughout the period of exposure of the experiment. The temperature was maintained at 22 ° C±2°C.
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (1500Lux).
3. The pH of the control cultures needs to be noted during the study and the pH of the control medium should not increase by more than 1.5 units during the test.
4. The orbital shaking incubator was set at a speed of 120 revolutions per minute throughout the study period. This is to provide constant shaking to the algal cells to keep them in suspension and to ensure that they do not settle down on the bottom of the test vessel.
5. Study duration : The experimental phase of the study was lasted for a period of 72 hours.
Reference substance (positive control):
not specified
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.726 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: calculated from equation through probit analysis
Details on results:
The microscopic observations were also noted in each of the experimental flasks. All the cells appeared healthy, round and green throughout the test duration in the control and in the experimental flask also no significant changes were observed.
Reported statistics and error estimates:
To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) was determined.

Table 1: Showing the average cell count using Haemocytometer of the test vessels at an

equal interval of 24hrs, 48hrs and 72hrs

Test vessels and test concentration

24 Hours

48 Hours

72 Hours

Control

Replicate1

15600

20800

26400

Replicate2

16400

20000

24000

Replicate3

16000

20400

24800

CAS No.526-75-0

0.5mg/l

Replicate1

13600

18400

23600

Replicate2

14000

16800

20000

1mg/l

Replicate1

12400

15600

19200

Replicate2

12800

15600

18000

2mg/l

Replicate1

12000

14400

17200

Replicate2

11600

14000

16800

4mg/l

Replicate1

12000

13200

14800

Replicate2

11600

12400

14000

8mg/l

Replicate1

11200

12000

13200

Replicate2

10400

10800

12000

16mg/l

Replicate1

10800

11200

12400

Replicate2

10400

10800

11200

  

 

Table 2: Showing the values of average specific growth rate and percentage inhibition

after an interval of 72 hours

 

CONTROL

6.25mg/l

12.5mg/l

25mg/l

50mg/l

100mg/l

200mg/l

Average

Specific

Growth rate (μ)

R1

0.32

R1

0.29

R1

0.22

R1

0.18

R1

0.13

R1

0.09

R1

0.07

R2

0.29

R2

0.23

R2

0.20

R2

0.17

R2

0.11

R2

0.06

R2

0.04

R3

0.30

 

Mean of Avg.

Specific

growth rate

0.31

0.26

0.21

0.18

0.12

0.08

0.05

Percentage

Inhibition (%I)

_

15.49

32.47

42.22

60.33

74.95

82.11

 


Table 3: Depicting pH values at 0 Hours and after 72 Hours of test item exposure to algae

Test vessels and

test concentration

0 Hours

72 Hours

CONTROL

Replicate1

6.68

6.71

Replicate2

6.67

6.77

Replicate3

6.68

6.78

Average

6.68

6.75

CAS No. 526-75-0

0.5mg/l

Replicate1

6.72

6.60

Replicate2

6.66

6.75

1mg/l

Replicate1

6.69

6.71

Replicate2

6.65

6.74

2mg/l

Replicate1

6.65

6.88

Replicate2

6.63

6.84

4mg/l

Replicate1

6.66

6.68

Replicate2

6.64

6.48

8mg/l

Replicate1

6.63

6.68

Replicate2

6.46

6.63

16mg/l

Replicate1

6.58

6.58

Replicate2

6.46

6.60

 

Validity criteria fulfilled:
yes
Conclusions:
Based on the growth inhibition of green alga Chlorella vulgaris by the test chemical 2,3-dimethylphenol (526-75-0), the EC50 was determine to be 2.726 mg/l.
Executive summary:

The study was designed to assess the toxic effects of the test compound 2,3-dimethylphenol (526-75-0) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance 2,3-dimethylphenol (526-75-0) was prepared by adding 4 mg of test item in 250 ml of BBM to get the final concentration of 16 mg/L. This stock solution was kept for stirring for 05 minutes to obtain a homogenous solution for the experiment. The test concentrations >0.5 mg/L, 1 mg/L, 2 mg/L, 4 mg/L, 8 mg/L, 16 mg/L were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item 2,3-dimethylphenol (526-75-0) to various nominal test concentrations, EC50 was determine to be 2.726 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was hazardous and can be consider to be classified aq aquatic chronic 2 as per the CLP classification criteria.

Description of key information

The study was designed to assess the toxic effects of the test compound 2,3-dimethylphenol (526-75-0) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).

Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance 2,3-dimethylphenol (526-75-0) was prepared by adding 4 mg of test item in 250 ml of BBM to get the final concentration of 16 mg/L. This stock solution was kept for stirring for 05 minutes to obtain a homogenous solution for the experiment. The test concentrations >0.5 mg/L, 1 mg/L, 2 mg/L, 4 mg/L, 8 mg/L, 16 mg/L were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution.

For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.

Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.

As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201

After 72 hours of exposure to test item 2,3-dimethylphenol (526-75-0) to various nominal test concentrations, EC50 was determine to be 2.726 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was hazardous and can be consider to be classified aq aquatic chronic 2 as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
2.726 mg/L

Additional information

Various studies for the target chemical 2,3-Dimethylphenol (CAS No. 526-75-0) were reviewed to summarize the following information:

In an experimental study, the study was designed to assess the toxic effects of the test compound 2,3-dimethylphenol (526-75-0) on the green alga Chlorella vulgaris. Test was conducted in compliance with the OECD guideline 201 (Alga, Growth Inhibition Test).Test was carried out in 100mL conical flasks which were carefully autoclaved and sterilized. The test solution in each of these test vessels was kept constant which is 60 ml so that a sufficient amount of head space was left. The test substance 2,3-dimethylphenol (526-75-0) was prepared by adding 4 mg of test item in 250 ml of BBM to get the final concentration of 16 mg/L. This stock solution was kept for stirring for 05 minutes to obtain a homogenous solution for the experiment. The test concentrations >0.5 mg/L, 1 mg/L, 2 mg/L, 4 mg/L, 8 mg/L, 16 mg/L were chosen according to the available data of the test item. The concentrations chosen were set up to the water solubility limit. The remaining test solutions were prepared by dilution from the above stock solution.For the assessment of algal growth, the test was conducted in replicates. The control flask was maintained in triplicates as recommended in the OECD guideline and the test concentration were selected in geometric series which were maintained in duplicates. To obtain a quantitative concentration-response relationship by regression analysis, a linearizing transformation of the response data into probit was performed. Using the same, effective concentration (EC) were determined.Algal growth was calculated daily by counting the cells microscopically with the help of haemocytometer. For microscopic observations the cultures were observed daily with the help of a microscope to verify a normal and healthy appearance of the algal culture and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test item). Apart from this, the cell count of each test vessel was also noted with the help of a microscope and haemocytometer. By spectrophotometer the absorbance values of each test vessel and control vessel was noted at 680nm.The BBM was taken as blank for both control and test vessels. The absorbance value of each vessel was in line with the average specific growth rate.As per OECD 201, the biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72 hr test period. This corresponds to a specific growth rate of 0.92 per day. Thus, the observed specific growth rate in the control cultures during the experiment was 0.358 per day. Secondly the mean coefficient of variation for section by section specific growth rates (days 0-1, 1-2 & 2-3, for 72 hr tests) in the control cultures must not exceed 35%. Thus, the observed mean coefficient of variation in the control cultures during the experiment was 33.42%. Thirdly the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 10%. Thus, the observed coefficient of variation of average specific growth rates during the experiment in control cultures was 8.26%. Hence, the test is considered valid as per OECD guideline, 201After 72 hours of exposure to test item 2,3-dimethylphenol (526-75-0) to various nominal test concentrations, EC50 was determine to be 2.726 mg/l graphically and through probit analysis. Based on the EC50, it can be concluded that the chemical was hazardous and can be consider to be classified aq aquatic chronic 2 as per the CLP classification criteria.

In another CRO lab experimental study report,aim of this study was to evaluate the nature of chemical test chemical 2,3-dimethylphenol (526-75-0) when comes in contact with the test organism Desmodesmus subspicatus (previous name: Scenedesmus subspicatus). The stock solution 100.0 mg/l was prepared by dissolving dark orange powder in OECD growth medium. Test solutions of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture.0,10,18,32,58,100 mg/l concentration were used.With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration EC50 was calculated using nonlinear regression by the software Prism 4.0. Effect on the growth of algae was determine after an exposure period of 72 hrs.The median effective concentration (EC50) for the test substance 2,3-dimethylphenol (526-75-0), in algae was determined to be 92.3 mg/l(95% CI:73.1-116.5) on the basis of growth rate inhibition effects in a 72 hour study. Based on the EC50 value, which indicates that the substance is likely to be hazardous to aquatic algae and can be classified as aquatic chronic 3 category as per the CLP classification criteria.

In an experimental study, toxicity of substance 2, 3-dimethyl phenol (CAS no. 526 -75 -0) to aquatic algae Selenastrum capricornutum assay was carried out using standard procedure for Algaltoxkit Fᵀᴹ (A. Kahru et. al; 2000). Selenastrum capricornutum was used as a test organism. The study was based on the effect on the test compound 2, 3-dimethyl phenol to Selenastrum capricornutum under static conditions at a temperature of 25°C for 72 hrs. The initial algal culture was prepared from the immobilized algal beads and the deimmobilized cells were pregrown in the sterile growth medium (25°C, 6000-8000 lux). Before the experiment the culture was diluted to OD670=0.001(corresponds toapprox.104cells/ml)and cultivated further for 4 days.The EC50 value of 2, 3-dimethyl phenol is determined to be 50mg/L in Selenastrum capricornutum. Thus, based on the EC50 value, substance 2, 3-dimethyl phenol can be considered as toxic to aquatic organisms. Since the chemical is readily biodegradable in nature, chemical 2, 3-dimethyl phenol can be considered as non-toxic to aquatic organisms and thus can be considered to be not classified as per the CLP classification criteria.

Even though varied results for short term toxicity of aquatic invertebrate were available but, based on the harmonised classification in ECHA ,we can consider that the test chemical is toxic and can be classified as aquatic chronic 2.