Piperazine

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 - 3 April 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

0.2 mL of the test solution was collected from each test container in each test group at the start of the exposure and before water change (24 hours after the start of exposure), and the volume was adjusted to 100 mL with purified water before analysis by LC/MS. The test substance concentration of each test solution was quantified from the ratio with the peak area of the standard solution.

Vehicle:

no

Details on test solutions:

Preparation of test solution.
The dilution water was at the time of preparing the test solution, aerated before preparation and kept at 20 ± 1 ° C in a constant temperature bath.
The stock solution for generating the test solutions was prepared by dissolving 500 mg of test item in 500 mL dilution water (without solvent).
500 mL of each test solution was prepared by diluting the required amount of the test item stock solution to obtain the following test concentrations: 10.0, 21.0, 44.0, 94.0 and 200 mg/L, 100 mL of these solutions was placed in 4 test containers per concentration.
Dilution water without adding the test substance was used as the control group.
The state (appearance) of the test solution at the time of preparation was colorless and transparent in all test groups.

Test concentration setting  
Based on the results of the preliminary test (2 tests, 10 daphnids/ test concentration) shown in the table below, the concentration of this test was determined as follows.
Test concentrations for final test : Control group, 10.0, 21.0, 44.0, 94.0, 200 mg / L (ratio 2.1)

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organism
1) Japanese name : Daphnia magna
2) Scientific name : Daphnia magna
3) Obtained from: National Institute for Environmental Studies, Environmental Agency (currently National Institute for Environmental Studies)
4) Data of obtain : 18 July 1995
5) Growth stage: Female juvenile (within 24 hours)
6) Breeding conditions for parent Daphnia to obtain test organisms:

Feeding water: Dilution water (Elendt see 3.2)
Feeding density: Below 1 daphnid/ 80 mL (25 daphnids/ 2L)
Feeding container: 2 L glass container
Water temperature: 20 ± 1℃
Dissolved acid concentration: more than 60% saturation concentration
pH:6.7~8.5
illumination: Indoor light, 16 hours bright (under 800 lux) / 8 hours dark

During the feeding period: From 4 March 2003 to 1 April 2003
Parental mortality 2 weeks prior to exposure： 0%
Occurrence of dormant eggs and males: None
Food type: Chlorella vulgaris (Unicellular green algae) (Centrifugate the algae culture solution and replace it with diluted water before use)
Feeding amount: 0.2 mg C (Organic carbon content) / head / day
Breeding water exchange: Replace regularly (3 times / week). Remove juveniles daily.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19.5 - 20.4 °C measured in all test solutions at the start and end

pH:

8.1 - 10.2 measured in all test solustions at the start and end

start End
Control 8.3 8.1
10.0 9.0 8.8
21.0 9.3 9.1
44.0 9.6 9.4
94.0 9.9 9.7
200 10.2 10.0

Dissolved oxygen:

8.5 - 8.8 mg/L measured in all test solutions at the start and end

Salinity:

Elendt test medium

Nominal and measured concentrations:

Results of specific chemical analysis presented in Table 1:
Nominal: Control group, 10.0, 21.0, 44.0, 94.0 and 200 mg/L (ratio 2.1)
Measured at start (0 hour): <0.2, 11, 19.9, 48.3, 95.4 and 194 mg/L (95 - 110% recovery)
Measured just before medium change (24 hours): <0.2, 9.4, 17.5, 35.8, 93.5 and 183 mg/L (81 - 99% recovery)
Geomean: -, 10.2, 18.7, 41.6, 94.4 and 188 mg/L

Details on test conditions:

3. Test method
3.1. Test conditions:
1) Exposure method: Semi-stop type (replace the entire amount of test solution after 24 hours) Cover the water surface with a Teflon sheet
2) Exposure period: 48 hours
3) Test solution volume: 100 mL/container
4) Number of consecutive: 4 containers/test concentration
5) Number of tested organisms: 20 daphnids/ test concentration (5 daphnids/container)
6) Test temperature: 20 ± 1 ℃
7) Dissolved oxygen concentration: more than 60% saturation concentration
8) pH: Do not adjust the pH of the test solution
9) Lighting: Indoor light, 16 hours bright (under 800 lux) / 8 hours
10) Feeding: None

3.2. Diluted water
  Elendt M4, the prepared water described in OECD Chemical Test Guideline No. 211 "Daphnia magna breeding test", was used. The composition is shown in illustration field

3.3. Test container, constant temperature bath, etc.
1} Test container: 100 mL glass beaker (cover the water surface with a Teflon sheet)
2) Constant temperature bath: PVC water tank (constant temperature device, made by Taitec, Cool Knit CL-80F type)
3) Coolant temperature gauge: Yokogawa Electric 2455 02 type No.1
4) Dissolved oxygen meter: Electrochemical instrument DOL-10 type No.2
5) pH meter: HM-40V type No.1 manufactured by Toa Denpa Kogyo
6) pH meter: Desktop pH meter made by East Asia Radio Industry, HM-40V No. 2
7} Thermometer： TNA-120 made by Tasco Japan
8} Illuminance meter: IM-2D made by Topcon

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

123 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 94 - 200 mg/L

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

106 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 87.1 - 128 mg/L

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

105.4 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 86.6 - 127.2 mg/L

Details on results:

As a result of the specific chemical analysis of the test solutions the concentration of the test substance used to calculate the inhibitory concentration was determined using the nominal test concentrations.

Results with reference substance (positive control):

48-hour half-Immobilisation concentration (EiC50) = 0.60 mg/L (95% confidence interval 0.52 to 0.69 mg/L) by reference substance (potassium dichromate, special grade reagent)
This value is almost the same as the EiC50 value (n = 9 since June 1998) at our institute.
Mean ± standard deviation = 0.77 ± 0.18 mg/L
Minimum value to maximum value = 0.57 - 1.02 mg/L

Reported statistics and error estimates:

Probit method and Calculate 95% confidence intervals if possible.

Preliminary test results 

First time			Second time
Concentration (mg/L)	Immobilisation rate (%)		Concentration (mg/L)	Immobilisation rate (%)
	After 24 hours	After 48 Hours		After 24 hours	After 48 Hours
Control	0	0	Control	0	0
1.00	0	0	5.00	0	0
10.0	0	0	110	40	80
100	20	30	230	100	100
1000	100	100	500	100	100

Semi-static exposure

The water temperature was 20 ± 1 ° C in all test groups, and the dissolved oxygen concentration was more than 60% of the saturated dissolved oxygen concentration (saturated dissolved oxygen concentration at 20.0 °C: 8.8 mg/L) in all test groups. Meting the criteria. The pH was higher in the concentration group (8.8 to 10.2) than in the control group (8.1 to 8.3). This is considered to be due to the test substance.

Table 2 The number of Immobilized Daphnia Magna (percent Immobility)

Nominal	Cumulative Number of Immobilized Daphnia (%)
Concentration (mg/L)	24 hours	48 hours
Control	0 (0)	0 (0)
10.0	0 (0)	0 (0)
21.0	0 (0)	0 (0)
44.0	0 (0)	1 (5)
94.0	3 (15)	5(25)
200	20 (100)	20 (100)

 

Validity criteria fulfilled:

yes

Remarks:

In the controls no daphnids were immobilised and the dissolved oxygen concentration at the end of the test should was >8 mg/L in control and test vessels.

Conclusions:

The test substance is described in detail
The test species and culturing conditions are described in detail
The test conditions and test medium are described in detail
The test results are presented in detail
The exposure is well characterized

Executive summary:

The acute ecotoxicity of piperazine to daphnia magna has been tested according to OECD TG 202 under GLP conditions.

The final test concentration range (Control, 10.0, 21.0, 44.0, 94.0 and 200 mg/L) was selected based on two range finding tests. The test was performed semi-statically by refreshing the test solution after 24hours. The exposure concentrations were monitored using specific chemical analysis applying LC/MS with a LOD of 0.2 mg/L. All test concentrations remained withing 20% of the nominal test concentration at both sampling times and therefore the nominal test concentration was used for the quantification of the endpoints.

The 24h EC50 is 123 mg/L (95% CI: 94 - 200 mg test item/L)

The 48h EC50 is 106 mg/L (95% CI: 87.1 - 128 mg test item/L

The test substance had a purity of 99.4%

Corrected for active ingredient content the 48h EC50 is 105.4 mg/L (95% CI: 86.6 - 127.3 mg/L)

Description of key information

There are three short-term toxicity to aquatic invertebrates tests available. Two tests were performed with the freshwater species Daphnia magna and one with the marine species Acartia tonsa.

The exposure concentrations during the test were however only monitored in the study with Daphnia magna (Mitsubishi, 2003). This study is therefore considered to be the Key study. The test substance was observed to be stable during the test and therefore the nominal test concentration was used for the calculation of the endpoint.

The 48h LC50 of piperazine for daphnia is observed to be 105.4 mg/L (95% CI: 86.6 - 127.2 mg/L)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

LC50

Effect concentration:

105.4 mg/L