2-bromoethylammonium bromide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Experimental test result performed using standard OECD test guidelines

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Principles of method if other than guideline:

This study was designed to assess the effect of the test item 2-bromoethanaminium bromide (CAS No. 2576-47-8) on the growth of green alga Chlorella vulgaris. The study was conducted in accordance with “OECD guideline for testing of chemicals No. 201 – Alga, growth inhibition test”.

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): 2-bromoethanaminium bromide
- Molecular formula (if other than submission substance): C2H6BrN.BrH
- Molecular weight (if other than submission substance): 204.892 g/mol
- Smiles notation (if other than submission substance): C(C[NH3+])Br.[BrH-]
- InChI: 1S/C2H6BrN.BrH/c3-1-2-4;/h1-2,4H2;1H
- Substance type: Organic
- Physical state: solid
- Purity: 100.01% (argenometry)
- Storage condition of test material: Ambient Temp (23 to 27 °C). Hygroscopic in nature.
- Other: Stable in recommended storage conditions
- Solubility in water: 84020.07 mg/l

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test solution was prepared in aseptic condition. The test item 2-bromoethylammonium bromide was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 2 hours 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 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
- Strain:Chlorella vulgaris
- Source (laboratory, culture collection): The fresh water green alga Chlorella vulgaris, was used as the test system (organism). Sterile, unicellular, suspension cultures of algae were obtained from National Environmental Engineering Research Institute (NEERI), Nagpur and maintained at Unique Ecotox Research Laboratory, Nagpur. The culture was examined under the microscope to confirm that it was unicellular, healthy and not contaminated.
- 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

Total exposure duration:

72 h

Test temperature:

22 °C ±2°C

pH:

6.8 ±0.3

Nominal and measured concentrations:

Six test concentration were: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: Conical flasks
- Type (delete if not applicable): No data available
- Material, size, headspace, fill volume: Conical flasks of 100 ml size was used for the study.
- Aeration: No data available
- Type of flow-through (e.g. peristaltic or proportional diluter): No data available
- Renewal rate of test solution (frequency/flow rate): No data available
- Initial cells density: 29.16 x104 cells/mL
- Control end cells density: No data available
- 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
- No. of vessels per vehicle control (replicates): No data available

GROWTH MEDIUM
- Standard medium used: No data available
- 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: No data available
- Photoperiod: 16 Hour Light Period : 8 Hour Dark Period
- Light intensity and quality: continuous, uniform fluorescent illumination(1500Lux)
- Salinity (for marine algae): No data available

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 available
- Other: 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.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: All the six concentrations were in geometric series spaced by a factor of 2.
- Justification for using less concentrations than requested by guideline: No data available
- Range finding study: No data available
- Test concentrations: Six test concentration were: 6.25mg/l,12.5mg/l,25mg/l,50mg/l,100mg/l,200mg/l (Nominal concentrations)
- Results used to determine the conditions for the definitive study: No data available

6.1. Test solution: The test solution was prepared in aseptic condition. The test item 2-bromoethylammonium bromide was prepared by adding 50 mg of test item in 250 ml of BBM to get the final concentration of 200 mg/L. This stock solution was kept for stirring for 2 hours 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 of the culture was kept 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.

6.2. Test vessels: All the tests were 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 60ml so that a sufficient amount of head space was left.

6.3. Replicates: For the assessment of algal growth, the study was conducted in replicates. The control vessel was maintained in triplicates as recommended in the OECD guideline and the test concentrations were selected in geometric series which were maintained in duplicates.

6.4. Incubation:
i) 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.
ii) The test vessels were incubated with a continuous, uniform fluorescent illumination(1500Lux).
iii) 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.
iv) 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.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

129.652 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Control vessels: The microscopic observations were noted down in each of the control vessel. All the cells appeared healthy, round and green throughout the study duration in the control. Also, the drift in pH in the control vessels did not increase by >1.5 units when observed on 72 hours as compared to 0 hours. The average pH drift observed in the control vessels was 0.11 units.

Test vessels: The effect of the test item on the green algae Chlorella vulgaris culture was observed at nominal test concentration of >6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L, 100 mg/L,
200 mg/L. All the six concentrations were in geometric series spaced by a factor of 2. The microscopic observations were also noted in each of the test vessel. All the cells appeared
healthy, round and green throughout the study duration and no significant changes were observed up to the concentration of 200 mg/l. EC50 was found to be 129.652 mg/l graphically through probit analysis.

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
Replicate 1	20000	36400	46400
Replicate 2	20800	37200	47200
Replicate 3	22800	38800	47200
CAS No. 2576 -47 -8
6.25 mg/l
Replicate 1	22800	26000	27600
Replicate 2	22800	25200	26000
12.5 mg/l
Replicate 1	22800	23600	26000
Replicate 2	21200	22800	25200
25 mg/l
Replicate 1	20400	21200	25600
Replicate 2	18800	20800	24000
50 mg/l
Replicate 1	18000	18800	23200
Replicate 2	18000	17200	23600
100 mg/l
Replicate 1	16400	18000	22800
Replicate 2	18000	15600	20400
200 mg/l
Replicate 1	16400	16000	20400
Replicate 2	15200	15600	21600

 

 

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	0.511	R1	0.338	R1	0.318	R1	0.313	R1	0.280	R1	0.274	R1	0.237
	R2	0.517	R2	0.318	R2	0.308	R2	0.291	R2	0.286	R2	0.237	R2	0.256
	R3	0.522
Mean of Avg. Specific growth rate	0.517		0.328		0.313		0.302		0.283		0.256		0.247
Percentage Inhibition (%I)	_		36.497		39.429		41.500		45.214		50.470		52.212

 

 

 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
Replicate 1	6.60	6.68
Replicate 2	6.61	6.74
Replicate 3	6.62	6.75
Average	6.61	6.72
CAS No. 2576 -47 -8
6.25 mg/l
Replicate 1	6.58	6.46
Replicate 2	6.59	6.44
12.5 mg/l
Replicate 1	6.58	6.46
Replicate 2	6.59	6.42
25 mg/l
Replicate 1	6.54	6.38
Replicate 2	6.53	6.39
50 mg/l
Replicate 1	6.49	6.39
Replicate 2	6.48	6.29
100 mg/l
Replicate 1	6.42	6.29
Replicate 2	6.43	6.28
200 mg/l
Replicate 1	6.30	6.26
Replicate 2	6.31	6.25

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test item 2-bromoethylammonium bromide (CAS No. 2576-47-8) to various nominal test concentrations, EC50 was found to be 129.652 mg/l graphically and through probit analysis.

Executive summary:

The effect of test item 2-bromoethylammonium bromide, CAS No. 2576-47-8 was studied on the growth of fresh water green alga Chlorella vulgaris. The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 129.652 mg/L. Thus based on this value, it can be concluded that the substance can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Description of key information

The effect of test item 2-bromoethylammonium bromide, CAS No. 2576-47-8 was studied on the growth of fresh water green alga Chlorella vulgaris (UERL study report, Sustainability Support Services (Europe) AB, 2017). The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 129.652 mg/L. Thus based on this value, it can be concluded that the substance 2-bromoethylammonium bromide can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

129.65 mg/L

Additional information

Experimental key studyfor the target chemical 2-bromoethanaminium bromide (CAS no. 2576-47-8) which is supported further by total 2 studies (both from secondary source) for its closest structurally similar read across substance with logKow as the primary descriptor were reviewed for the toxicity to aquatic algae and cyanobacteria end point which are summarized as below:

 

In an experimental key study, the effect of test item 2-bromoethylammonium bromide, CAS No. 2576-47-8 was studied on the growth of fresh water green alga Chlorella vulgaris(UERL study report, Sustainability Support Services (Europe) AB, 2017). The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25 mg/L,12.5 mg/L,25 mg/L,50 mg/L,100 mg/L,200 mg/L. The test concentrations were prepared using stock solution of the test item using mineral media. The green alga was exposed to the test concentration for a period of 72 hours to observe average specific growth rate and % growth inhibition under the effect of the test item. EC50 calculated graphically through probit analysis was observed to be 129.652 mg/L. Thus based on this value, it can be concluded that the substance 2-bromoethylammonium bromide can be considered as non-toxic to aquatic organisms and thus cannot be classified as hazardous as per the CLP classification criteria.

 

In a supporting studyof read across substance Glutamic acid (CAS no. 56-86-0) from peer secondary source (OECD SIDS Initial Assessment Report, CoCam 4, 2013),short term toxicity to Pseudokirchnerella subcapitata (algae) study was carried out for 72 hrs. The study was performed according to OECD Guideline 201 (Alga, Growth Inhibition Test). The study was based on the effects of the read across compound Glutamic acid on Pseudokirchnerella subcapitata in a static fresh water system at an average temperature of 23 ± 2ᵒC. Range finding study was carried out at a test conc. of 100 mg/l, respectively. Test chemical conc. used for the study were1, 3, 10, 31 and 100, respectively. The control group was prepared for this study. The concentration of the test substance during the exposure period was within +/-20% of the nominal concentrations. Therefore, all test results were determined as the nominal concentrations. In the control group, the concentration of the test substance was not detected. Pseudokirchnerella subcapitata was used as test organism obtained from American Type Culture Collection (ATCC. U.S.A.). Pre-culture, algal suspensions were added to replicate flasks containing the test substance solutions and adjusted to an algal biomass concentration of 1x104 cells/mL. Flasks were incubated at 23 +/-2℃under continuous illumination (4440 -8880 Lux) and constantly shaken at approximately 100 rpm using a shaker incubator for a period of 72 hours. Flasks were rotated upward and to the left at 24 hours intervals during incubation in an attempt to equally distribute light intensity. Six replicate flasks were prepared for each dosed group and control group. All test and control cultures were inspected microscopically at 72 hours. At 72 hours after initiation of exposure, the percent inhibition of growth in the dosed group and control group was calculated and a concentration-percent inhibition growth curve generated. The ErC50 (0 -72 hours) and EyC50 (0 -72 hours) were calculated and their 95% confidence limits were statistically analyzed using the Logit method. Statistical analysis was conducted using SAS (version 9.1.3). For determination of NOEC and LOEC values, in the case of the multiple comparisons for the determination of the NOEC and LOEC for average specific growth rate and yield, Bartlett's test was conducted to test for homogeneity. One-way analysis of variance (ANOVA) was employed on homogeneous data. And then, if significant, followed by Dunnett's t-test for multiple comparisons. A Kruskal-Wallis test was employed on heterogeneous data and if significant, followed by Steel test for multiple comparisons. Statistical analysis was conducted using SAS (version 9.1.3). The 72hr-EC50 values of L-glutamic acid (CAS No. 56 -86 -0) to Pseudokirchneriella subcapitata was determined as 68.5 mg/L (95% confidence limits: 62.4 -74.1 mg/L) and as 54.4 mg/L (95% confidence limits: 50.0 -59.4 mg/L), respectively. The NOEC and LOEC for the results of the average specific growth rate and yield were determined as 31 mg/L and as 100 mg/L, respectively. Thus, based on EC50 value, it can be concluded that the substance Glutamic acid can be considered as toxic to aquatic organisms. Since the test chemical is readily biodegradable in nature, chemical Glutamic acid can be considered as non-toxic to aquatic organisms and thus can be considered as not classified as per the CLP classification criteria.

Another supporting study of read across substance11 -Aminoundecanoic acid (CAS no. 2432-99-7) on Pseudokirchneriella subcapitata in a static fresh water system was carried out for 72 hrs (OECD SIDS Initial Assessment Report, SIAM 15, 2002).The study was performed according to OECD Guideline 201 (Alga, Growth Inhibition Test) and other method C3 described in directive 92/69/EEC of the European Commission. The study was based on the effects of the test compound 11 -Aminoundecanoic acid on Pseudokirchnerella subcapitata in a static fresh water system at an average temperature of 23.0 ± 1ᵒC and pH of 8.0, respectively under constantly illumination of light with intensity between 6000 to 10000lx. Test chemical conc. used for the study were 0, 4.5, 7.4, 12.3, 20.2, 33.4, 55.1, 90.9 and 150 mg/l, respectively (nominal concentrations). Stock solution prepared at the beginning of the test, by adding 150 ml of substance in 1 l of dilution water. After about one hour of agitation, the solution was and homogenate. The No Observed Effect Concentration (NOEC) is determined by a statistical procedure: analysis of variance and Dunnett's test. The 72hr-EC50 values of 11 -Aminoundecanoic acid (CAS No. 2432 -99 -7) to Pseudokirchneriella subcapitata was determined as 53 and 23 mg/L, respectively for growth rate and biomass and the NOEC value was determined to be 4.5 mg/l, respectively. Thus, based on EC50 value, it can be concluded that the substance 11 -Aminoundecanoic acid can be considered as toxic to aquatic organisms. Since the test chemical is readily biodegradable in nature, chemical 11 -Aminoundecanoic acid can be considered as non-toxic to aquatic organisms and thus can be considered as not classified as per the CLP classification criteria.

 

Thus, based on the overall reported results for target chemical 2 -bromoethanaminium bromide (UERL study report, 2017) and for its read across substance (from secondary source), it can be concluded that the test substance 2 -bromoethanaminium bromide can be considered as non-toxic to aquatic organisms at environmentally relevant concentrations and can be considered to be not classified as per the CLP classification criteria.