2,3-xylenol

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10 mg/L, 18 mg/L, 32.4 mg/L, 58.32 mg/L and 105 mg/L
- Sampling method:
- Sample storage conditions before analysis: sample was analysed immediately after sampling

Vehicle:

no

Details on test solutions:

The saturated test solution was prepared by dissolving 500mg of test chemical in 500ml of OECD media, and allowed for stirring for 96 hours, which was then filtered and the final saturated stock solution obtained was 123.55 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Length: 8 – 14 μm
- Weight: 2 - 3 μm
- Source: Sterile, unicellular, suspension cultures of algae were obtained from the laboratory for Biological Research in Aquatic Pollution (LABRAP) at the University of Ghent in Belgium and maintained in
Laboratory.
- Method of cultivation: OECD medium

ACCLIMATION
- Culturing media and conditions (same as test or not): The medium to be used for the growth of algae was OECD medium.
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25°C

pH:

At 0 Hour: control 7.40-7.74; At 72 hour control: 7.70-7.87;
At 0 in test concentrations: 7.48-7.56; At 72 hours: 7.40-7.74

Nominal and measured concentrations:

Test chemical concentrations used for the study were contorl, 10 mg/L, 18 mg/L, 32.4 mg/L, 58.32 mg/L and 105 mg/L , respectively.

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): Three replicates for each test concentration
- No. of vessels per control (replicates): Three replicates for Control

GROWTH MEDIUM
- Standard medium used: yes, OECD medium was used as a test medium in the study.

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(7000-8000Lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell counts were measured using heamocytometer under microscope.
- 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 1.8.
- Test concentrations: Six test concentration were: 0, 10 mg/L, 18 mg/L, 32.4 mg/L, 58.32 mg/L and 105 mg/L (Nominal concentrations)
- Results used to determine the conditions for the definitive study: cell growth 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 25 ° C
2. The test vessels were incubated with a continuous, uniform fluorescent illumination (7000-8000Lux).
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):

yes

Remarks:

Potassium dichromate (K2Cr2O7) was used as a reference substance.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

57.5 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, sickle shaped and green throughout the test duration in the control.

Results with reference substance (positive control):

- Results with reference substance valid?
- EC50: 0.868 mg/l

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: Assessment of test concentrations

Sr. No	Concentrations mg/L	Wavelength (nm)	Absorbance	Temperature (°C)
1	blank	270	0.00	25°C
2	3.75	270	0.0396	25°C
3	7.50	270	0.0774	25°C
4	15.00	270	0.1327	25°C
5	30.00	270	0.2800	25°C
6	45.00	270	0.4182	25°C
7	60.00	270	0.5739	25°C
8	75.00	270	0.7043	25°C
9	90.00	270	0.8553	25°C
10	105.00	270	0.9887	25°C
11	120.00	270	1.1326	25°C
12	135.00	270	1.2760	25°C
13	150.00	270	1.3983	25°C

 

The absorbance and concentrations were recorded at270 nm.

 

Concentration After Analytical Determination

 

		0 Hours	0 Hours	72 Hours	72 Hours
SR. No	concentrations (mg/L)	Absorbance (mean)	Analytical concentrations	Absorbance (mean)	Analytical concentrations
1	control	0.00	0.00	0.00	0.00
2	10mg/L	0.1	10.41	0.10	10.72
3	18mg/L	0.18	19.14	0.17	17.93
4	32.4mg/L	0.32	33.87	0.31	32.92
5	58.32mg/L	0.56	59.11	0.54	57.61
6	105mg/L	1.00	106.51	1.00	106.65

pH AND TEMPERATURE

 

Test Concentration(mg/L)	Experimental Flasks	pH		Temperature °C
		0 Hours	72 Hours	0 Hours	72 Hours
control	R1	7.74	7.87	25	25
control	R2	7.70	7.70	25	25
control	R3	7.40	7.74	25	25
10mg/L	R1	7.52	7.74	25	25
10mg/L	R2	7.49	7.70	25	25
10mg/L	R3	7.48	7.72	25	25
18mg/L	R1	7.47	7.63	25	25
18mg/L	R2	7.54	7.53	25	25
18mg/L	R3	7.55	7.56	25	25
32.4mg/L	R1	7.56	7.40	25	25
32.4mg/L	R2	7.42	7.61	25	25
32.4mg/L	R3	7.49	7.60	25	25
58.32mg/L	R1	7.52	7.58	25	25
58.32mg/L	R2	7.50	7.80	25	25
58.32mg/L	R3	7.54	7.71	25	25
105mg/L	R1	7.53	7.65	25	25
105mg/L	R2	7.51	7.65	25	25
105mg/L	R3	7.59	7.71	25	25

The pH was measured at the beginning of the test and after 72 hr of exposure. The pH of the control medium did not increase by more than 1.5 units during the test.

 

Experimental Flasks and Test Concentration(mg/L)	0 Hr Cell Count	24 Hr Cell Count	48 Hr Cell Count	72 Hr Cell Count	Avg Specific Growth Rate (µ)	Mean Avg Specific Growth Rate (µ)	Percent Inhibition(%)
control	10000	30000	90000	310000	1.14	1.16	-
control	10000	35000	85000	350000	1.19
control	10000	30000	75000	325000	1.16
10mg/L	10000	25000	50000	325000	1.16	1.14	1.98
10mg/L	10000	25000	50000	315000	1.15
10mg/L	10000	35000	40000	280000	1.11
18mg/L	10000	30000	60000	215000	1.02	1.03	11.54
18mg/L	10000	25000	45000	245000	1.07
18mg/L	10000	20000	40000	200000	1.00
32.4mg/L	10000	20000	40000	140000	0.88	0.83	28.99
32.4mg/L	10000	20000	35000	110000	0.80
32.4mg/L	10000	20000	40000	110000	0.80
58.32mg/L	10000	15000	25000	75000	0.67	0.59	49.28
58.32mg/L	10000	10000	20000	60000	0.60
58.32mg/L	10000	10000	15000	45000	0.50
105mg/L	10000	10000	20000	30000	0.37	0.30	74.14
105mg/L	10000	10000	15000	25000	0.31
105mg/L	10000	10000	15000	20000	0.23

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth inhibition of green alga Pseudokirchneriella subcapitata by the test chemical, the 72 hr median effect concentration (EC50) was determined to be 57.5 mg/l (calculated from equation through probit analysis).

Executive summary:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 250mg of test chemical in 250ml of OECD media, to get at concentratino of 1000 mg/l , which was then filtered and the final saturated stock solution obtained was 989.8 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 7000 -8000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 57.5 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Description of key information

Short term toxicity to Algae:

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 250mg of test chemical in 250ml of OECD media, to get at concentratino of 1000 mg/l , which was then filtered and the final saturated stock solution obtained was 989.8 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 7000 -8000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 57.5 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

57.5 mg/L

Additional information

Experimental studies of the test chemical and various supporting weight of evidence studies for its structurally similar read across chemical were reviewed for toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

A freshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Pseudokirchneriella subcapitata. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. OECD medium composed of macronutrients, micronutrients, alkaline EDTA solution and iron solution was used as a growth medium. The saturated test solution was prepared by dissolving 250mg of test chemical in 250ml of OECD media, to get at concentratino of 1000 mg/l , which was then filtered and the final saturated stock solution obtained was 989.8 mg/L, verified analytically by UV-Vis Spectrophotometer. Further, exposure concentrations of 0, 0.25, 0.5, 1, 2, 4 and 8 mg/l, respectively was from the saturated test concentrations. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 21 to 24 ± 2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 7000 -8000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. The cultures were counted and observed daily with the help of a microscope. Potassium dichromate (K2Cr2O7) was used as a reference substance. The 72 hr EC50 value of the reference substance was determined to be 0.868 mg/l. The biomass in the control vessel have increased exponentially by a factor of 16 and the mean coefficient of variation of specific growth rate was not exceeded 35%, thus fulfilling the validity criterion. As the concentration of the test chemical being tested has been satisfactorily maintained within ± 20 % of the nominal concentration throughout the test. Therefore, the analysis of the results was based on nominal concentration. On the basis of growth rate of the test organism, the 72 hr median effect concentration (EC50) was determined to be 57.5 mg/l (calculated from equation through probit analysis). On the basis of this value, chemical was considered as toxic to aquatic algae and hence, considered to be classified in 'aquatic chronic category 3' as per the CLP classification criteria.

In an experimental study from study report (2017),afreshwater algal growth inhibition test was conducted for 72 hrs for assessing the effect of test chemical on green algae Chlorella vulgaris. The test was performed in accordance to OECD guideline No. 201 – Alga growth inhibition test under static condition. Initial cell density of the culture was kept at 10000 cells/ml. 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. The test solution was prepared in aseptic condition. The test chemical was prepared by adding 4 mg of test chemical in 250ml of BBM to get the final concentration of 16 mg/L. 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 10000 cells/ml. Green algae were exposed to nominal concentration of test chemical (0, 0.5, 1, 2, 4, 8 and 16 mg/l) in 100 ml conical flasks. Test vessel were placed in orbital shaking incubator for 72 hrs at a room at a temperature of 22±2°C under a photoperiod of 16:8 hr light: dark conditions and with a continuous uniform illumination of 3000-4000 lux light intensity, respectively. The speed of the orbital shaking incubator was set at a 120 revolutions per minute throughout the study period. Control containing medium without test chemical was used for the study. The cultures were counted and observed daily with the help of an automated cell counter to verify a normal and healthy appearance of the algae cells and also to observe any abnormal appearance of the algae (as may be caused by the exposure of the test chemical). As per the OECD guideline No. 201 – Alga growth inhibition test, the biomass in the control cultures have increase exponentially by a factor of at least 16 within the 72 hr test period, the mean coefficient of variation by section specific growth rate in the control cultures not exceeded 35% (i.e., reported as 12.66%) and the coefficient of variation of average specific growth rate during the whole test period in replicate control cultures was not exceeded 10% (i.e., reported as 5.27%), thus, fulfilling the validity of the criteria. All the cells appeared healthy, round and green throughout the study duration in the control and no significant changes were observed up to the concentration of 16 mg/l. On the basis of growth rate of the test organism Chlorella vulgaris, the 72 hrs median effect concentration (ErC50) value was determined to be 2.726 mg/l.

 

In a supporting weight of evidence study, toxicity to aquatic algae study was conducted for 72 hrs for assessing the effect of test chemical on green algae. The test was performed in accordance to OECD Guideline 201 (Alga, Growth Inhibition Test) in a static system. Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) of strain 86.81 SAG obtained from Institute of botany of the ASCR with an initial biomass conc. 5000 cells /ml was used as a test organism. The stock solution 100 mg/l was prepared by dissolving test chemical 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. Test chemical concentrations were not verified analytically. Nominal test chemical concentrations used for the study were 0, 10, 18, 32, 58 and 100 mg/l, respectively. Desmodesmus subspicatus were exposed to test chemical in 50 ml glass vessel in a volume of 15 ml of liquid solution containing both the chemical and media. Control solution vessel containing OECD medium without the test chemical was also setup during the study. The beakers were placed in a room at a temperature of 23±2°C with a continuous light intensity of 6000-8000 lx, respectively. Alongwith the test chemical, one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. Cell counting was carried out using microscope with counting chamber Cyrus I or electronic particle counter. ErC50 was calculated using non linear regression by the software Prism 4.0. In the control test vessel containing OECD growth medium without test chemical, the coefficient of variation of average growth rate in replicates during the whole test period was 1.7% (in control). The 24 hr EC50 value of the reference substance was determined to be 0.77 mg/l. On the basis of the effect of test chemical on the growth rate of the test organism Desmodesmus subspicatus, the 72 hr EC50 value was determined to be 92.3 mg/l (95% C. I. 73.1 to 116.5 mg/l) (nominal concentration).

 

Another toxicity to aquatic algae study was carried out for assessing the effect of test chemical (from A. Kahru et. al., 2000). The bioassays were performed according to the standard operational procedures of the Toxkits (Algaltoxkit FTM). Algaltoxkit FTM follows the standard protocol as specified in the OECD TG 201 and ISO test method respecitvely. Test chemical was dissolved in deionized water. Selenastrum capricornutum green algae) was used as a test organism. 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 to approx. 10000 cells/ml) and cultivated further for 4 days. Study was performed under static conditions for 72 hrs at 25°C under a test condition with light intensity of 6000 to 8000 lux. The inhibition of the growth rate of algae Selenastrum capricornutum was measured. On the basis of the effect of test chemical on growth rate of the test algae, the 72 hr EC50 was determined to be 50 mg/l.

 

For the test chemical from secondary source (2012), toxicity to aquatic algae study was carried out for assessing the effect of test chemical. The study was performed in accordance with the OECD Guideline 201 (Alga, Growth Inhibition Test). Pseudokirchneriella subcapitata (green algae) ATCC22662 obtained from American Type Culture Collection was used as a test organism. 200mg of the test substance was dissolved with dilution water to produce a stock solution of 1000 mg/L . Test solution was prepared in each test vessel by mixing the appropriate volume of the stock solution with dilution water.  Test chemical concentrations were verified analytically by HPLC-UV instrument. For the analytical determnations, samples were diluted with pure water. Thus, test chemical concentrations used for the study were  0, 5.6, 10, 18, 32, 56 and 100 mg/l (nominal concentrations) and 0, 5.41, 9.64, 17.5, 31.1, 54.2 and 96.8 mg/l (measured conc.), respectively. Test algae was exposed with the test chemical in an 300 ml erlenmeyer flask with silicon rubber plug (air permeability). Standard OECD medium was used as a test medium for the study. Test organism was exposed for a period of 72 hr and at test conditions of 23°C temperature, pH 8.8-9.1 (at the start of exposure) and 8.1-10.4 at the end of exposure under a continuous photoperiod with light intensity of 4000-5000 Lx. Dilution water was used for the control group. All experiment was performed in triplicates. Algal cells were counted with a particle counter at 24, 48, and 72 h. Microscopic observation were done at 72 h. The pH of the test solution was measured at the start, and the end of the exposure. The temperature in the incubator and light intensity were measured once a day during the exposure. Potassium dichromate was used as a reference substance. The 72 hr EbC50 value of the reference substance potassium dichromate was determined to be 0.65 mgl. On the basis of the effect of test chemical on growth rate of the test algae, the 72 hr NOEC and EC50 value was determined to be 32 and >100 mg/l (nominal conc.). On the basis of effect on biomass, the 72 hr NOEC and EC50 value was determined to be 31.1 and 55.6 mg/l (measured (arithmetic mean) conc.), respectively.

 

In a supporting weight of evidence study from authoritative database, toxicity to aquatic algae study was carried out for assessing the effect of test chemical. The study was performed following the principles of OECD Guideline 201 (Alga, Growth Inhibition Test) under static conditions. Based on the effect of test chemical on growth rate of the test algae, the 72 hr NOEC and EC50 value was determined to be 1.8 and 9.7 mg/l. On the basis of effect on AUG, the 72 hr NOEC and EC50 value was determined to be 2.0 and 6.7 mg/l, respectively.

 

On the basis of the above results, it can be concluded that the test chemical was considered to be toxic to aquatic algae and hence, considered to be classified in aquatic chronic category 3 as per the CLP classification criteria.