Tetrabutylammonium hydroxide

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 according to the guideline.

Qualifier:

according to guideline

Guideline:

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 250 ml of BBM to get the final concentration of 100 mg/L. This stock solution was kept for stirring for 10 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test chemical.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM
- Common name: Green alga
- 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:

Nominal test concentrations: 3.125mg/l, 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l and 100mg/l respectively. 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: 3.125mg/l, 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l and 100mg/l respectively (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):

no

Duration:

72 h

Dose descriptor:

EC0

Effect conc.:

200 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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: No effect was observed

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	24 Hours	48 Hours	72 Hours
test concentration
Control
Replicate1	13200	14000	18400
Replicate2	11600	14000	18000
Replicate3	12800	15600	19600
Test chemical
3.125mg/l
Replicate1	10400	12800	17200
Replicate2	12400	14800	18000
6.25mg/l
Replicate1	10800	13600	17200
Replicate2	12400	15600	17600
12.5mg/l
Replicate1	14000	14800	16000
Replicate2	14000	15600	18400
25mg/l
Replicate1	10800	14000	18000
Replicate2	10400	10800	16000
50mg/l
Replicate1	12400	14000	18000
Replicate2	11600	14800	15600
100mg/l
Replicate1	12000	14800	17600
Replicate2	11200	12800	14800

Table 2: Showing the values of average specific growth rate and percentage inhibition after an interval of 72 hours

	CONTROL		3.125mg/l		6.25mg/l		12.5mg/l		25mg/l		50mg/l		100mg/
Average Specific Growth rate (μ)	R1	0.203	R1	0.180	R1	0.180	R1	0.156	R1	0.195	R1	0.195	R1	0.188
	R2	0.195	R2	0.195	R2	0.188	R2	0.203	R2	0.156	R2	0.148	R2	0.130
	R3	0.224
Mean of Avg. Specific growth rate	0.207		0.188		0.184		0.179		0.176		0.172		0.159
Percentage Inhibition (%I)	-		9.373		11.175		13.410		15.173		17.203		23.227

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

Test vessels and	0 Hours	72 Hours
test concentration
CONTROL
Replicate1	6.82	7.06
Replicate2	6.83	7.06
Replicate3	6.79	7.07
Average	6.81	7.06
Test chemical
3.125mg/l
Replicate1	6.68	6.72
Replicate2	6.67	6.69
6.25mg/l
Replicate1	6.72	6.75
Replicate2	6.70	6.72
12.5mg/l
Replicate1	6.75	6.76
Replicate2	6.72	6.72
25mg/l
Replicate1	6.77	6.72
Replicate2	6.92	6.77
50mg/l
Replicate1	6.87	6.85
Replicate2	6.92	6.91
100mg/l
Replicate1	7.04	6.95
Replicate2	6.97	7.02

Validity criteria fulfilled:

yes

Conclusions:

Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determine to be >200 mg/l as no effects were observed upto the concentration of 200 mg/l. Thus on the basis of these values, chemical was considered as non-toxic to algae and hence, considered to be ‘’not classified’’ as per the CLP classification criteria.

Executive summary:

An algal growth inhibition study was conducted for 72 hrs on green algae. The test was performed in accordance with the‘’OECD Guideline 201 (Alga, Growth Inhibition Test)’’. Chlorella vulgaris was used as a test organism. The test solution was prepared in 250 ml of BBM to get the final concentration of 100 mg/L. This stock solution was kept for stirring for 10 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test chemical. 3.125mg/l, 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l and 100mg/l  respectively nominal test concentrations were prepared. All the six concentration were in geometric series spaced by a factor of 2. Test conducted under the static system. 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. 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 was considered to be valid as per OECD guideline, 201. Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determine to be >200 mg/l as no effects were observed upto the concentration of 200 mg/l. Thus on the basis of these values, chemical was considered as non-toxic to algae and hence, considered to be ‘’not classified’’ as per the CLP classification criteria.

Description of key information

An algal growth inhibition study was conducted for 72 hrs on green algae. The test was performed in accordance with the‘’OECD Guideline 201 (Alga, Growth Inhibition Test)’’. Chlorella vulgaris was used as a test organism. The test solution was prepared in 250 ml of BBM to get the final concentration of 100 mg/L. This stock solution was kept for stirring for 10 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test chemical. 3.125mg/l, 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l and 100mg/l  respectively nominal test concentrations were prepared. All the six concentration were in geometric series spaced by a factor of 2. Test conducted under the static system. 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. 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 was considered to be valid as per OECD guideline, 201. Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determine to be >200 mg/l as no effects were observed upto the concentration of 200 mg/l. Thus on the basis of these values, chemical was considered as non-toxic to algae and hence, considered to be ‘’not classified’’ as per the CLP classification criteria.

Additional information

Toxicity to aquatic algae and cyanobacteria:

Data available for the test chemicals and structually and functionally similar read across chemicals have been reviewed to determine the toxicity of the test chemical on aquatic algae.The studies are as mentioned below:

An algal growth inhibition study was conducted for 72 hrs on green algae. The test was performed in accordance with the‘’OECD Guideline 201 (Alga, Growth Inhibition Test)’’. Chlorella vulgaris was used as a test organism. The test solution was prepared in 250 ml of BBM to get the final concentration of 100 mg/L. This stock solution was kept for stirring for 10 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test chemical. 3.125mg/l, 6.25mg/l,12.5mg/l, 25mg/l, 50mg/l and 100mg/l  respectively nominal test concentrations were prepared. All the six concentration were in geometric series spaced by a factor of 2. Test conducted under the static system. 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. 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 was considered to be valid as per OECD guideline, 201. Based on the growth rate inhibition of green alga Chlorella vulgaris by the test chemical, the EC50 was determine to be >200 mg/l as no effects were observed upto the concentration of 200 mg/l. Thus on the basis of these values, chemical was considered as non-toxic to algae and hence, considered to be ‘’not classified’’ as per the CLP classification criteria.

Above study further supported by the second study from experimental source. Principle of this study was to evaluate the nature of test chemical when comes in contact with the test organism. Test was conducted according to the OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 100 mg/l was prepared in OECD growth medium. Test conducted under the static system for 72 hours on limit test concentration of 100 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of test chemical, effect concentration the differences in the mean of control and sample were estimated by the t-test for independent groups at a 95% confidence level, all individual replicates were used (STATISTICA CZ - data analysis software system, version 9.0, StatSoft, Inc) Statistically significant differences are for p < 0.05. Effects on the growth rate of algae was determined after an exposure period of 72 hrs. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical for 72 hours, only 2.1% inhibition was observed at limit test concentration of 100 mg/l. Thus on the basis of % inhibition, EC50 was consider to be > 100 mg/l. Based on the % inhibition value, test chemical was consider likely to be nonhazardous to aquatic algae and cannot be classified as per the CLP classification criteria.

Similar study was conducted to assess the effect of test chemical on green algae. Test was conducted according to the OECD Guideline 201 (Alga, Growth Inhibition Test). Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) was used as test organism. The stock solution 160 mg/l was prepared in OECD growth medium. Test solution of required concentrations were prepared by mixing the stock solution of the test sample with OECD growth medium and inoculum culture. Test conducted under the static system and tested at the various nominal concentrations 0, 10, 20, 40, 80 and 160 mg/l. With the test substance one positive control Potassium dichromate (K2Cr2O7) was also run simultaneously. After the exposure of chemical, effect concentration ErC50 was calculated using nonlinear regression by the software Prism 4.0. Growth rate inhibition of Desmodesmus subspicatus were used a key parameter to determine the effect of chemical on algae by providing exposure period of 72 hours. Based on the growth rate inhibition of algae Desmodesmus subspicatus (previous name: Scenedesmus subspicatus) due to the exposure of test chemical for 72 hours, the ErC50 value was determined to be 259.5 mg/l with the 95% CI of 226.7 mg/l to 297.5 mg/l. Thus on the basis of ErC50 value, test chemical consider to be nontoxic and not classified as per the CLP classification criteria.

On the basis of above all studies and effects observations from various sources, it was concluded that the test chemical was non toxic and not classified as per the CLP classification criteria.