Diphenylacetic acid

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 Diphenylacetic acid (CAS No. 117-34-0) 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): Diphenylacetic acid
- Molecular formula (if other than submission substance): C14H12O2
- Molecular weight (if other than submission substance): 212.247 g/mol
- Smiles notation (if other than submission substance): C(c1ccccc1)(c1ccccc1)C(O)=O
- InChI: 1S/C14H12O2/c15-14(16)13(11-7-3-1-4-8-11)12-9-5-2-6-10-12/h1-10,13H,(H,15,16)
- Substance type: Organic
- Physical state: solid
- Storage condition of test material: 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.) Hygroscopic in nature.
- Other: Stable in recommended storage conditions
- Solubility in water: 873.0825 mg/l

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

The test solution was prepared in aseptic condition. The test item diphenyl acetic acid 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 diphenyl acetic acid 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.:

> 200 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 >200 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. 117 -34 -0
6.25 mg/l
Replicate 1	13600	27600	36400
Replicate 2	16800	29200	38000
12.5 mg/l
Replicate 1	15200	27200	36400
Replicate 2	14400	26400	34800
25 mg/l
Replicate 1	13200	24400	34000
Replicate 2	13200	23600	33200
50 mg/l
Replicate 1	10800	28400	32400
Replicate 2	11600	26800	30800
100 mg/l
Replicate 1	13200	24000	29600
Replicate 2	11600	22800	28000
200 mg/l
Replicate 1	8800	19600	27600
Replicate 2	8400	20400	26000

 

 

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	0.430	R1	0.430	R1	0.407	R1	0.391	R1	0.361	R1	0.338
	R2	0.517	R2	0.445	R2	0.415	R2	0.399	R2	0.374	R2	0.343	R2	0.318
	R3	0.522
Mean of Avg. Specific growth rate	0.517		0.437		0.423		0.403		0.383		0.352		0.328
Percentage Inhibition (%I)	_		15.352		18.186		21.901		25.872		31.855		36.497

 

 

 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. 117 -34 -0
6.25 mg/l
Replicate 1	6.48	6.23
Replicate 2	6.47	6.26
12.5 mg/l
Replicate 1	6.45	6.29
Replicate 2	6.48	6.30
25 mg/l
Replicate 1	6.46	6.32
Replicate 2	6.47	6.33
50 mg/l
Replicate 1	6.32	6.34
Replicate 2	6.30	6.34
100 mg/l
Replicate 1	6.29	5.81
Replicate 2	6.28	5.78
200 mg/l
Replicate 1	6.25	4.87
Replicate 2	6.24	4.89

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test item diphenyl acetic acid (CAS No. 117-34-0) to various nominal test concentrations, EC50 was found to be >200 mg/l graphically and through probit analysis.

Executive summary:

The effect of test item diphenyl acetic acid, CAS No. 117-34-0 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 >200 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 diphenyl acetic acid, CAS No. 117-34-0 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 >200 mg/L. Thus based on this value, it can be concluded that the substance diphenyl acetic acid 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:

200 mg/L

Additional information

Experimental key study for the target chemical Diphenylacetic acid (CAS no. 117-34-0) which is supported further by total 2 studies (both from peer reviewed journal) 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 diphenyl acetic acid, CAS No. 117-34-0 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 >200 mg/L. Thus based on this value, it can be concluded that the substance diphenyl acetic acid 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 study of read across substanceBenzoic acid (CAS no. 65-85-0) from peer reviewed journal (GLENN W. STRATTON & CHARLES T. CORKE, 1982), short term toxicity study was carried out using cyanobacteria (blue-green algae) Anabaena inaequalis, A. cylindrica, and A.variabilis, and the green algae Chlorella pyrenoidosa and Scenedesmus quadricauda as a test organism. The study was based on the effects of the test compound Benzoic acid on algae in a static fresh water system. Stock solutions of test chemical were prepared in pesticide grade acetone. The proper level of acetone (0.1 %v/v) to use in each bioassay system was determined using the solvent-pesticide interaction analysis technique. Chemical was tested at a minimum of five concentrations ranging from 0 to 100 mg/l (nominal concentration). Test organisms used for the study include (blue-green algae) Anabaena inaequalis, A. cylindrica, and A.variabilis, and the green algae Chlorella pyrenoidosa and Scenedesmus quadricauda, respectively.

Cultures of Anabaena inaequalis and A. cylindrica were maintained in a liquid nitrogen-free medium at a temperature of 20°C and a light intensity of 7000 lux on a 12 h light-dark cycle whereas the other cultures such as A.variabilis, Chlorella pyrenoidosa and Scenedesmus quadricauda were maintained under the same conditions except the growth medium was supplemented with 1.5 g NaNO3/l. Photosynthesis was assayed by following the uptake of 14CO2 from NaH14CO3 (Amersham/Searle, Oakville, Ontario, Canada). Plastic tissue culture flasks with a total internal volume of 74 ml were employed as assay chambers. Each contained 9.9ml of cell suspension (containing 6-5 × 10(4)cyanobacteria or 1.0 × 10(5)green algal cells/ml), 0.1 ml of radioisotope (to give a final activity of 0.1µCi/ml) and 0.01 ml of test chemical. The flasks were incubated for 3 h and photosynthetic activity was assayed. Per cent inhibition values were calculated relative to photosynthetic activity in control systems (solvent only) and EC50 values determined by probit or regression analysis, where applicable. Analyses for significant differences (p = 0.05) were performed using Dunnett's testand Duncan's multiple range test. Based on the effect of Benzoic acid on the photosynthesis of the test organisms, the EC50 value was determined to be 5, 60, 55, 60 and 75 mg/l for Anabaena inaequalis, A. cylindrical, A.variabilis, Chlorella pyrenoidosa and Scenedesmus quadricauda respectively. Thus, based on this value, it can be concluded that the test chemical Benzoic acid can be considered as toxic to aquatic organisms. Since the test chemical is readily biodegradable in nature, chemical Benzoic 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 ofthe same read across substanceBenzoic acid (CAS no. 65-85-0) on Pseudokirchneriella subcapitata in a static fresh water system was carried out for 48 hrs. The study was performed according to closed system algal toxicity test. Algal inoculum was withdrawn from the chemostat operated under a steady state, and transferred into 300mL BOD bottles, together with dilution water (with growth medium) and toxicants. The BOD bottles were filled completely, leaving no headspace. A water seal was provided to ensure a closed test environment. The bottles were then placed on an orbital shaker operated at 100 rpm. Temperature and light intensity were kept at 24±1◦C and 65 µEm2/s (± 10%), respectively. The final yield and algal growth rate based on cell density counts was determined. The population density of the algae was determined using an electronic particle counter. Probit analysis was applied to determine the concentration– response relationship and the median effective concentration (EC50). One-tail Dunnett’s procedure was applied for the estimation of NOEC and LOEC values at 5% level of significance. On the basis effect on growth rate and yield of the test organism Pseudokirchneriella subcapitata (green algae), the 48 hr EC50 value was determined to be 83.29 and 36.39 mg/l, respectively and the NOEC value was determined to be 4.81 and 9.62 mg/l, respectively. Thus, based on this value, it can be concluded that the test chemical Benzoic acid can be considered as toxic to aquatic organisms. Since the test chemical is readily biodegradable in nature, chemical Benzoic 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 Diphenylacetic acid (UERL study report, 2017) and for its read across substance (from peer reviewed journal), it can be concluded that the test substance Diphenylacetic acid 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.