Tripiperazine dicitrate

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:

according 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 Tripiperazine dicitrate; CAS No. 144-29-6 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:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Piperazine citrate / tripiperazine dicitrate- Molecular formula: C12H30N6•Cl2H16O14 - Molecular weight: 642.76 g/mole - Substance type: Organic - Physical state: Solid

Analytical monitoring:

yes

Details on sampling:

not specified

Vehicle:

yes

Details on test solutions:

Test solution: The test solution was prepared in aseptic condition. The test item Tripiperazine dicitrate was prepared by adding 50mg of test item in 250ml of BBM to get the final concentration of 200mg/L. This stock solution was kept for stirring for 20 minutes to obtain a homogenous solution for the experiment. The test concentrations were chosen according to the available data of the test item. 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 1 X 104cells/ml. Care was taken to have a homogeneous solution for the experiment.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.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.

Test organisms (species):

Chlorella vulgaris

Details on test organisms:

TEST ORGANISM- Common name:green alga - Strain:Chlorella vulgaris- Source (laboratory, culture collection):laboratoryACCLIMATION- 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

Hardness:

No data

Test temperature:

24 °C ±2°C.

pH:

6.2-7.5

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM- Test vessel: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.- Material, size, headspace, fill volume:100ml- No. of vessels per concentration (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.- No. of vessels per control (replicates):Three replicates for Control- No. of vessels per vehicle control (replicates):Two replicates for each test concentrationGROWTH MEDIUM- Standard medium used: yesOTHER TEST CONDITIONS- Sterile test conditions: yes- Adjustment of pH:6.8 ±0.3 - Photoperiod:16 Hour Light Period : 8 Hour Dark Period- Light intensity and quality:White Fluorescent Light with intensity 1500LuxEFFECT PARAMETERS MEASURED (with observation intervals if applicable) :- Determination of cell concentrations: spectrophotometerTEST CONCENTRATIONS- Spacing factor for test concentrations:2

Reference substance (positive control):

not specified

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

87.912 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: graphically and through probit analysis

Details on results:

Test concentrations: The effect of the test item Tripiperazine dicitrate on the green algae Chlorella vulgaris culture was observed at nominal test concentration of 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100 mg/l and 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 in the control and in the test vessels black dotted rings were observed in concentrations of 100mg/l and 200mg/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 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	49 x 10^4	70 x 10^4	104.5 x 10^4
Replicate 2	59 x 10^4	78 x 10^4	97 x 10^4
Replicate 3	54 x 10^4	73.5 x 10^4	101.5 x 10^4
CAS No. 144-29-6
6.25mg/l
Replicate 1	48.5 x 10^4	38 x 10^4	36.5 x 10^4
Replicate 2	43.5 x 10^4	42.5 x 10^4	36.5 x 10^4
12.5mg/l
Replicate 1	43 x 10^4	35.5 x 10^4	32 x 10^4
Replicate 2	42 x 10^4	37 x 10^4	34 x 10^4
25mg/l
Replicate 1	37 x 10^4	33.5 x 10^4	28 x 10^4
Replicate 2	37 x 10^4	32 x 10^4	29 x 10^4
50mg/l
Replicate 1	30.5 x 10^4	26 x 10^4	24 x 10^4
Replicate 2	26 x 10^4	23 x 10^4	22.5 x 10^4
100mg/l
Replicate 1	22 x 10^4	14.5 x 10^4	10 x 10^4
Replicate 2	24 x 10^4	11 x 10^4	8 x 10^4
200mg/l
Replicate 1	13 x 10^4	8 x 10^4	4.5 x 10^4
Replicate 2	12 x 10^4	9.5 x 10^4	4 x 10^4

 

 

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	1.550	R1	1.199	R1	1.155	R1	1.111	R1	1.059	R1	0.768	R1	0.501
	R2	1.525	R2	1.199	R2	1.175	R2	1.122	R2	1.038	R2	0.693	R2	0.462
	R3	1.540
Mean of Avg. Specific growth rate	1.538		1.199		1.165		1.117		1.049		0.730		0.482
Percentage Inhibition (%I)	_		22.046		24.240		27.411		31.831		52.521		68.683

 

 

 

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	6.7	7.4
Replicate 2	6.8	7.6
Replicate 3	6.8	7.5
CAS No. 144-29-6
6.25mg/l
Replicate 1	6.8	7.3
Replicate 2	6.8	7.4
12.5mg/l
Replicate 1	6.8	7.4
Replicate 2	6.7	7.4
25mg/l
Replicate 1	6.7	7.2
Replicate 2	6.7	7.3
50mg/l
Replicate 1	6.6	7.1
Replicate 2	6.6	7.2
100mg/l
Replicate 1	6.5	7.0
Replicate 2	6.5	7.0
200mg/l
Replicate 1	6.2	6.7
Replicate 2	6.2	6.6

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to test item Tripiperazine dicitrate (CAS No. 144-29-6) to various nominal test concentrations, EC50 was found to be 87.912mg/l graphically and through probit analysis.

Executive summary:

The effect of test itemTripiperazine dicitrate, CAS No. 144-29-6 was studied on the growth of fresh water green algaChlorella vulgaris.The study was conducted following OECD guideline 201- Alga, growth inhibition test. The test concentration chosen for the study were 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/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 87.912mg/l. Thus, based on the EC50 value, test substance can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 3 but since the substance is readily biodegradable and the partition coefficient (log Kow) of the test chemical is also not ≥ 4 (i.e, reported in range -1.15), test chemical can be considered as non-hazardous to aquatic organisms as per the CLP criteria.

Description of key information

After 72 hours of exposure to test item Tripiperazine dicitrate (CAS No. 144-29-6) to various nominal test concentrations, EC50 was found to be 87.912mg/l graphically and through probit analysis. Thus, based on the EC50 value, test substance can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 3 but since the substance is readily biodegradable and the partition coefficient (log Kow) of the test chemical is also not ≥ 4 (i.e, reported in range -1.15), test chemical can be considered as non-hazardous to aquatic algae and cyanobacteria as per the CLP criteria.

Key value for chemical safety assessment

EC50 for freshwater algae:

87.912 mg/L

Additional information

Experimental key study for the test chemical Piperazine citrate (CAS No. 144-29-6) and supporting study for its structurally related substance were reviewed to summarize the following information for toxicity to aquatic algae endpoint:

In key study, the effect of test item Tripiperazine dicitrate, CAS No. 144-29-6 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 6.25mg/l, 12.5mg/l, 25mg/l, 50mg/l, 100mg/l and 200mg/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 87.912mg/l.Thus, based on the EC50 value, test substancecan be considered astoxic to aquatic organisms and thus can be classified as aquatic chronic category 3but since the substance is readily biodegradable and the partition coefficient (log Kow) of the test chemical is also not ≥ 4 (i.e, reported in range -1.15), test chemical can be considered as non-hazardous to aquatic algae and cyanobacteria as per the CLP criteria.

Supporting above study, as per J-check database short term toxicity to algae study was carried out for 72 hrs for the structurally related substance piperazine (CAS 110 -85 -0). The study was performed according to OECD Guideline 201 (Alga, Growth Inhibition Test). On the basis of growth rate and AUG of the test organism algae,the 72 hrs EC50 values was determined to be 130 and 91 mg/l, respectively and NOEC values was determined to be 34 and 46 mg/l, respectively.Thus, based on the EC50 value (91 mg/l), it can be concluded that the substance can be considered as toxic to aquatic organisms and thus can be classified as aquatic chronic category 3but since the read across substance is readily biodegradable, it can be considered as non-hazardous to aquatic organisms as per the CLP criteria.

Based on the overall reported results for target and read across substance, it can be concluded that the substance Piperazine citrate can be considered as non hazardous to aquatic algae and cyanobacteria as per the CLP criteria.