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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 using standard OECD test guidelines
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Principles of method if other than guideline:
This study was designed to assess the effect of 4-nitro-o-toluidine; CAS No.99-52-5 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:
- IUPAC name 2-methyl-4-nitroaniline
- Name of test material: 4-nitro-o-toluidine
- Molecular formula: C7H8N2O2
- Molecular weight: 152.152 g/mol
- Physical state: Solid
- SMILES: c1(cc(c(N)cc1)C)[N+](=O)[O-]
- InChI: 1S/C7H8N2O2/c1-5-4-6(9(10)11)2-3-7(5)8/h2-4H,8H2,1H3
- Physical appearance: Yellow powder
- Stability: Stable in recommended storage conditions
- Storage conditions: Ambient Temp (23 to 27 °C) (Keep container tightly closed in a dry & well-ventilated place. Store in cool place. Do not expose to air, light & moisture.)
- Water solubility: 202.17 mg/l
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
The test solution was prepared in aseptic condition. The test item 4-nitro-o-toluidine 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 30 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 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: 0.5mg/l,1mg/l,2mg/l,4mg/l,8mg/l,16mg/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: 22.73 x104 cells/mL
- Control end cells density: No data available
- No. of organisms per vessel: 10000 cells/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: 0.5mg/l,1mg/l,2mg/l,4mg/l,8mg/l,16mg/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 4-nitro-o-toluidine 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 30 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 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.:
1.503 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 0.09 units.

Test vessels: The effect of the test item on the green algae Chlorella vulgaris culture was observed at nominal test concentration of >0.5 mg/L, 1 mg/L, 2 mg/L, 4 mg/L, 8 mg/L, 16 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 16 mg/l. EC50 was found to be 1.503 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

16800

32800

50000

Replicate 2

17200

35200

52400

Replicate 3

18000

36800

55600

CAS No. 99 -52 -5

0.5 mg/l

Replicate 1

18000

17200

14800

Replicate 2

16400

17200

16000

1 mg/l

Replicate 1

17200

18000

15600

Replicate 2

15600

18000

14000

2 mg/l

Replicate 1

14000

18400

13200

Replicate 2

13600

16400

14000

4 mg/l

Replicate 1

11600

15200

14000

Replicate 2

11600

16400

12400

8 mg/l

Replicate 1

10800

14800

10800

Replicate 2

11200

12400

12000

16 mg/l

Replicate 1

11600

13200

10800

Replicate 2

10400

14800

10400

 

 

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

 

CONTROL

0.5mg/l

1mg/l

2mg/l

4mg/l

8mg/l

16mg/l

Average Specific Growth rate (µ )

R1

0.536

R1

0.130

R1

0.148

R1

0.092

R1

0.112

R1

0.025

R1

0.025

 

R2

0.552

R2

0.156

R2

0.112

R2

0.112

R2

0.071

R2

0.060

R2

0.013

 

R3

0.571

 

Mean of Avg. Specific growth rate

0.553

0.143

0.130

0.102

0.091

0.043

0.019

Percentage Inhibition (%I)

_

74.041

76.477

81.507

83.390

92.192

96.501

 

 

 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

6.58

6.70

Replicate 2

6.59

6.68

Replicate 3

6.62

6.68

Average

6.6

6.69

CAS No. 99 -52 -5

0.5 mg/l

Replicate 1

6.62

6.57

Replicate 2

6.63

6.68

1 mg/l

Replicate 1

6.63

6.46

Replicate 2

6.64

6.58

2 mg/l

Replicate 1

6.68

6.59

Replicate 2

6.66

6.64

4 mg/l

Replicate 1

6.65

6.67

Replicate 2

6.66

6.70

8 mg/l

Replicate 1

6.63

6.77

Replicate 2

6.64

6.68

16 mg/l

Replicate 1

6.66

6.68

Replicate 2

6.65

6.69

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
After 72 hours of exposure to test item 4-nitro-o-toluidine (CAS No. 99-52-5) to various nominal test concentrations, EC50 was found to be 1.503 mg/l graphically and through probit analysis.
Executive summary:

The effect of test item 4-nitro-o-toluidine, CAS No. 99-52-5 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 0.5 mg/L,1 mg/L, 2mg/L, 4 mg/L, 8 mg/L,16 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 1.503 mg/L.Thus based on this value, it can be concluded that the substance can be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.

Description of key information

The effect of test item 4-nitro-o-toluidine, CAS No. 99-52-5 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 0.5 mg/L,1 mg/L, 2mg/L, 4 mg/L, 8 mg/L,16 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 1.503 mg/L.Thus based on this value, it can be concluded that the substance can be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:
1.503 mg/L

Additional information

An experimental key and supporting study for the target compound 2 -methyl-4 -nitroaniline (CAS No. 99 -52 -5) were reviewed to summarize the following information:

 

In an experimental key study, the effect of test item 4-nitro-o-toluidine, CAS No. 99-52-5 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 0.5 mg/L,1 mg/L, 2mg/L, 4 mg/L, 8 mg/L,16 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 1.503 mg/L.Thus based on this value, it can be concluded that the substance can be considered as toxic to aquatic organisms and thus can be considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.

 

In a supporting study from peer reviewed journal(Janet Riedl et. al; 2007), short term toxicity to Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus) study was carried out for 72 hrs. The study was performed according to OECD Guideline 201 (Alga, Growth Inhibition Test) and other method ISO 8692, respectively. The study was based on the effects of the test compound 2 -methyl-4 -nitroaniline (CAS no. 99 -52 -5) on Desmodesmus subspicatus in a static fresh water system at a temperature of 23 ± 2°C and pH of 6.9 ± 0.2, respectively. Test chemical concentration used for the study was 1000 mg/l (0.1%). Test solution was prepared by dissolving the chemical 2 -methyl-4 -nitroaniline in DMSO.Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus) was used as a test organism. Stock culture and pre-culture were prepared according to OECD Guideline 201 (Alga, Growth Inhibition Test). Liquid cultures of the unicellular green algae Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus) were grown photoautotrophically at 23 ± 2°C in an inorganic, sterilized medium with an additive of 1.5 mM NaHCO3 providing a final pH of the medium of 6.9 ± 0.2. Algae were incubated in a climatic chamber (HB 0714, Heraeus, under continuous light conditions from two types of fluorescent light tubes (L30W/830 Warmwhiteand L30W/77) providinga photosynthetic active radiation of 300 ± 20µE s-1m-2. The test was conducted in transparent microplates with 24 wells with an initial cell density of 5 × 103cells/ml, sealed with Parafilm M to minimize evaporation and shaken in intervals of 4 min with 100 rpm and 1 min pause. Incubation conditions were the same as used for cultivation. Compounds were tested on individual plates with 6 growth controls, 2 blank values without sample and inoculum and 16 sample concentrations. The growth inhibition was calculated by normalizing the data to the results of control cultures. Effect parameter population growth was determined by measuring the chlorophyll a content fluorometrically (SpectraMax Gemini EM, Molecular Devices, Sunnyvale, USA) after 0, 24, 48 and 72 h with an excitation wave length of 430 nm, emission of 690 nm and a cut-off filter <665 nm, evaluated with SoftMax Pro 3.1.1.The EC50 value of a compound was estimated by acquiring the best-fit concentration – response relationship using two fundamentally different models, namely either the Hill or the Weibull model, respectively. The concentration in the test vessels after an incubation time of 30 min and 72 h was verified by high performance liquid chromatography analysis. A 5lm particle size, reversed-phase C8 column (Lichrospher 60 RP select B, Merck, Darmstadt, Germany) was used as a stationary phase, the mobile phase was made up of methanol: water (40:60). The change in concentration over 72 h was analysed in bidistilled water, growth medium and algal suspension. Algae were centrifuged from the suspension before analysis. In principle, for each substance one concentration close to the EC50 value was investigated. For 2 -methyl-4 -nitroaniline no relevant loss of substance over the testing time could be determined (loss in exposure concentration of-0.7%). Based on effect on growth rate of the test organism Desmodesmus subspicatus, the 72 hr EC50 value was determined to be 9.7 mg/l, respectively. Thus, based on the EC50 value, it can be concluded that the substance 2 -methyl-4 -nitroaniline can be considered as toxic to aquatic organisms and thus can considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.

 

Thus, based on the overall reported results for target chemical 2-methyl-4-nitroaniline (fromUERL study reportand peer reviewed journal), it can be concluded that the test substance 2 -methyl-4 -nitroaniline can be considered as toxic to aquatic organisms and thus can considered to be classified in aquatic chronic category 2 as per the CLP classification criteria.