Piperazine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed according to GLP and guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

minor temperature increase

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical determinations were made on six occasions during the test. The concentrations were found to be stable within 20% of nominal concentrations.

Vehicle:

no

Details on test solutions:

Stock solutions dissolved in media and titrated to pH 7 with HCl, after dissolution

Test organisms (species):

Daphnia magna

Details on test organisms:

The animals used in the test were less than 24 hours old and were obtained from parent animals reproducing parthenogenically and having an age of 2-4 weeks (having previously produced at least one brood before use).
The test animals were taken from a Daphnia magna clone 4 stock, (origin: AquaSense, Kruislaan 411 a, PO Box 95125, 1090 HC Amsterdam) cultured in conformity with SOP E 1 (10).

Test type:

semi-static

Water media type:

freshwater

Total exposure duration:

21 d

Hardness:

13.2 - 14.4 °dH

Test temperature:

19.4 - 23.4°C. On two occasions temperature increased over the guideline recommendation. This is not thought to have affected the study

pH:

7.3 - 8.4

Dissolved oxygen:

7.6 - 9.3 mg/L

Nominal and measured concentrations:

Test concentrations: 0 mg/l, 3.1 mg/L, 6.25 mg/L, 12.5 mg/L, 25 mg/L, 50 mg/L.

Details on test conditions:

Culture animals were fed a diet of between 0.1 and 0.2 mg carbon per daphnid per day, in the form of the algal strain Chlorella vulgaris.

Deionized water: containing less than 10 µg/L copper with a conductivity of less than 5 µS and less than 2.0 mg/L NPOC-content.

Test-/Culturing medium: Reconstituted water (M4) according to Elendt (1990). The pH of this solution was 8.0 +/- 0.5 and the sum of Ca and Mg ions in the solution is 2.5 mmol/l with a molar ratio of Ca to Mg ions of approximately 4:1, and of Na to K 10:1. The dilution water was aerated before beeing used in the test.

Preparation of the stock solution: 1.11 g of the test substance was dissolved in 80 mL M4 and the solution was made up to 100 mL. The solution was agitated mechanically over night and the pH was adjusted to 8.0 with 1 M HCl. Test solutions were prepared by further dilution of the stock solution with M4.

The test vessels were 50 mLl glass beakers containing approximately 50 mL of the test medium. The test vessels were covered by glass plates during the tests.

The test solutions were renewed at least three times a week during the test. Saturation of test vessel walls with the test substance was undertaken.

10 replicate per test concentration and control. 1 Daphnid per replicate.

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: immobile neonates

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth

Details on results:

8. RESULTS
8.1 Preliminary test
A non-GLP 48 h acute toxicity test was performed at 0, 5, 11, 25, 55 and 120 mg/L using two replicates of 5 animals per concentration. No immobility was noted up to a nominal concentration of 120 mg/L. Due to the conflict between these results and a previous acute study also performed at Akzo Nobel (Balk & Meusen, 1989) where a 48 h EC50 of 26.1 mg/L was obtained, a second non-GLP 48 h preliminary test was performed at 0, 5, 11, 25, 55, 120 and 260 mg/L using only 5 daphnids per concentration. In this case one daphnid was immobilised at 55 and 120 mg/L and 3 daphnids at 260 mg/L.
A general observation was made that the daphnids were lethargic at higher concentrations (25 mg/L and above) and while not immobile, swimming behaviour was reduced compared to that of the controls.

8.2 Water quality and analytical results
Temperature measurements for new and old solutions at renewal min. 19.6; max 22.2°C.

Constant record of temperature over test time 19.4 to 23.4°C (see fig 3).

On two occasions the temperature increased over a period of 7 and 11 h, peaking at 22.3 and 23.4°C, respectively. Although these temperatures are above the 22°C recommendation in the OECD Guideline, these deviations from the protocol are not thought to have had any impact on the outcome of the study.

Oxygen concentration
min. 7.6; max. 9.3 mgO2/L
Results of Physico-chemical parameter measurements are presented in Appendix III
The test solutions were found to be stable over the test period. As concentrations were observed to be within ±20% of the nominals, all statistical evaluation has been based on nominal concentrations. A full description of the analytical method and results table is provided in Appendix IV.

Parent animal mortality
One daphnid died in each of the following concentrations: 3.1 (day 9), 6.25 (day 12), 25 (day 19), and 50 (day 10) mg/L. However, these mortalities were not considered to be concentration related. Five adults were found immobilised at 50 mg/L on day 19 of the study. They recovered prior to observations made on day 20.

Coefficient of variation of control fecundity
The number of juveniles per replicate in each concentration is shown in table 2. The validity criteria for the coefficient of variation (less than 25% in the control based on the number of living neonates for each parent animal alive at the end of the test) was reached.
The full data record of neonates released per day is presented in Appendix V (see any other information on results).

Statistical evaluation of the reproduction data and length and weight of parent animals at end of study.
The data was tested for normality using Chi-square test and found to be normally distributed. The data passed Hartley’s and Bartlett’s tests for homogeneity of variance (p<0.05). Analysis of variance was performed on the number of living neonates per parent using the Bonferroni t-test and verified with a second multiple comparison method, the Dunnett’s test (Appendix VI).

In both of these tests a significant effect (p<0.05) compared to the control was observed at 50 mg/L.

Further statistical analyses were performed using length and weight data.

Both length and weight data were found to be normally distributed and displayed homogeneity of variance. Multi-comparison tests of group weights and length of parent animals were employed. Animals exposed to the highest concentration were significantly different from the control (p<0.05).
Based on results from reproductive output and parental length, 50 mg/L can be taken as the Lowest Observed Effect Concentration (LOEC) and the No Observed Effect Concentration (NOEC) determined as 25 mg/L.
8.3 EC50 for parent animals
An EC50 based on reproduction capacity (or mortality) of parent animals at the end of the test could not be calculated due to insufficient parent mortality.
8.4 Any other biological effects observed
Two of the five broods released on day 15 at 25 mg/L were immobile, but not dead. At 50 mg/L all released broods throughout the test were immobile (but not dead). No other cases of neonate immobilisation were recorded at any concentration. On one occasion (day 19, on solution renewal) five of the parent daphnids at 50 mg/L were temporarily immobilised but all recovered and three of five continued to produce a final brood.
Immobilisation of released broods is not an official endpoint recommended by OECD Guideline 211. However, immobilisation of released neonates in the environment would certainly lead to their death and may be expected to affect population dynamics, so the authors consider that this endpoint can be employed without statistical support to generate the LOEC.

9. DISCUSSION
The concentrations used for the Daphnia reproduction test were selected by determining the 48 h EC50 for immobilisation and this is usually considered a good predictor of daphnid chronic toxicity. A previous acute study performed on daphnids (Balk & Meusen, 1989) provided an initial 48 h EC50 value of 26.1 mg/L, the lowest EC/LC50 value recorded of three acute studies carried out by these authors. The results of this first study were examined in detail and followed by two non-GLP preliminary studies prior to initiating the chronic test, During the preliminary studies the daphnids were observed to be less energetic than normal but they were capable of swimming and mortality was not recorded. Continuing the study for 7 days did not increase immobilisation. The authors of this report conclude that the lethargic behaviour of the daphnids exposed to high concentrations of piperazine was misinterpreted in the Balk & Meusen acute study as immobilisation. The first study also included mortality of the daphnids and while high immobilisation was reported at concentrations above 18 mg/L, high mortality (>25%) did not occur below 320 mg/L.
The chronic study supports this conclusion as concentration dependent mortality was negligible up to and including the highest concentration used (50 mg/L) over the 21 day test period. However, while these authors do not consider that lethargy is an acceptable substitute for immobilisation, some cases of genuine immobilisation were noted on one day of the study at 25 mg/L and at all brood releases at 50 mg/L. In this case the daphnids were clearly alive, moving their antennae but incapable of swimming while in the test solution. It is for this reason that the authors recommend that the statistically derived NOEC for reproduction of 25 mg/L is not acceptable in this case but a lower value of 12.5 mg/L be substituted, based on the observation of neonate immobilisation at 25 mg/L.

10. CONCLUSION
Data was found to be normally distributed and homogeneous. Using Dunnett’s and Bonferroni-t tests, the lowest Observed Effect Concentration (LOEC) based on reproductive output, weight and on parental body length was found to be 50 mg/L. Based on these statistical results the NOEC for reproduction, weight and length is 25 mg/L.
However, neonates from a brood released on day 15 were observed to be immobile (but not dead) in two vessels at 25 mg/L. This concentration is therefore taken as the LOEC and the NOEC is 12.5 mg/L.
The EC50 for adult mortality and for reproduction could not be determined due to insufficient mortality in any of the test concentrations.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

Statistical evaluation: the data (reproduction, length and weight of parent animals at the end of the study) was tested for normality using Chi-square test and found to be normally distributed. The data passed Hartley`s and Bartlett`s tests for homogeneity of variance (p < 0.05). Analysis of variance was performed on the number of living neonates per parent using the Bonferroni t-test and verified with a second multiple comparison method, the Dunnett`s test.

Appendix III: Tables 1 to 7

Table 1. Samples of “fresh” or “used” solutions analysed and day of sampling.

		Nominal concentration of sample (mg/L)
Solution	Day of study	FRESH	USED
1^stsolution	T0	0, 3.1, 50
1^stsolution	T2		0, 50
4^thsolution	T9	0, 3.1, 50
4^thsolution	T12		0, 3.1, 50
9^thsolution	T19	0, 50
9^thsolution	T21		0, 50

 

 

Table 2. Number of juveniles per parent alive at end of the test at each test concentration, mean number of neonates per concentration and coefficient of variance.

	Concentration (mg/L)
Rep no.	0	3.1	6.25	12.5	25	50
1	140	132	195	145	D	123
2	153	218	190	193	231	182
3	141	144	165	186	226	111
4	150	152	169	199	201	112
5	159	164	154	182	213	131
6	152	D	152	187	157	94
7	158	156	157	157	200	136
8	150	164	150	152	161	150
9	173	155	D	204	181	D
10	149	165	156	164	184	108
Mean	152.5	161.1	165.3	176.9	194.9	127.4
CV (%)	6.2	14.8	10.0	11.8	13.5	20.8

D = died during the test

Physico-chemical parameter measurements

 

Comparison of new (N) and old (O) values of control and highest concentration.

 

Table 3: pH-values

Nominal Test Conc.	N	O	N	O	N	O	N	O	N	O
[mg/L]	June 12	June 14	June 14	June 17	June 17	June 19	June 19	June 21	June 21	June 24
50	8.0	8.2	8.2	8.4	8.1	8.0	8.1	7.7	7.9	7.3
Control	8.0	8.1	8.2	7.9	8.1	7.7	8.1	7.5	7.9	7.3

 

Nominal Test Conc.	N	O	N	O	N	O	N	O
[mg/L]	June 24	June 26	June 26	June 28	June 28	July 01	July 01	July 03
50	8.1	7.5	7.8	7.7	8.0	7.3	8.4	7.7
Control	8.1	7.3	7.8	7.2	7.8	7.4	7.8	7.3

 

 

Table 4: Dissolved Oxygen Concentration [mg/l]

 

Nominal Test Conc.	N	O	N	O	N	O	N	O	N	O
[mg/L]	June 12	June 14	June 14	June 17	June 17	June 19	June 19	June 21	June 21	June 24
50	8.9	8.7	9.0	8.6	8.7	9.3	8.6	8.4	8.4	8.7
Control	8.7	8.7	8.9	8.8	8.9	9.1	8.6	8.1	8.5	7.6

 

Nominal Test Conc.	N	O	N	O	N	O	N	O
[mg/L]	June 24	June 26	June 26	June 28	June 28	July 01	July 01	July 03
50	8.6	8.6	8.7	8.5	8.7	8.0	8.9	8.5
Control	8.4	7.9	8.6	8.3	9.2	7.8	8.6	7.6

 

 

Table 5: Temperature [ºC]

 

Nominal Test Conc.	N	O	N	O	N	O	N	O	N	O
[mg/L]	June 12	June 14	June 14	June 17	June 17	June 19	June 19	June 21	June 21	June 24
50	21.5	20.9	20.9	22.0	21.0	20.4	20.2	20.4	20.6	19.9
Control	21.5	21.0	21.0	22.2	21.2	20.5	21.3	21.2	21.8	20.2

 

Nominal Test Conc.	N	O	N	O	N	O	N	O
[mg/L]	June 24	June 26	June 26	June 28	June 28	July 01	July 01	July 03
50	21.6	20.2	20.9	20.1	20.7	20.2	20.9	20.2
Control	21.6	20.1	20.9	19.9	19.6	20.6	20.8	20.3

 

  Table 6: Hardness ºdH]

 

Nominal Test Conc.	N	O	N	O
[mg/L]	June 12	June 14	July 01	July 03
50	13.9	13.2	14.1	14.4
Control	14.0	13.3	14.3	13.6

 

 

Table 7: Conductivity [s/cm]

 

Nominal Test Conc.	N	O
[mg/L]	June 21	June 24
50	699	701
Control	650	669

 

Results of chemical analysis

The results of analysis are shown in the table below.

Table 8. Nominal and measured concentrations and percentage of nominal

	Concentration (mg/l)
Day of study	Nominal	Measured	% of Nominal
T0	0	<LOQ	ND
	3.1	3.25	104
	50	48.08	96
T2	0	<LOQ	ND
	50	50.35	101
T9	0	<LOQ	ND
	3.1	3.26	105
	50	48.59	97
T12	0	<LOQ	ND
	3.1	2.88	93
	50	44.77	90
T19	0	<LOQ	ND
	50	48.95	98
T21	0	<LOQ	ND
	50	48.91	98

 

LOQ = Limit of Quantification

ND = Not Determined

Appendix V:Record of brood release, parental weight and length

 

CONTROL

 

Data	12.	13.	14.	15.	16.	17.	18.	19.	20.	21.	22.	23.	24.	25.	26.	27.	28.	29.	30.	01.	02.	03.
(2002)	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	07	07	07
Replicate No.	Number of juveniles																						Total
1	–	–	–	–	–	–	–	15	–	–	29	–	–	31	–	–	36	–	–	29	–	–	140
2	–	–	–	–	–	–	–	17	–	–	28	–	–	39	–	–	38	–	–	31	–	–	153
3	–	–	–	–	–	–	–	13	–	–	28	–	–	45	–	–	34	–	–	21	–	–	141
4	–	–	–	–	–	–	–	16	–	–	28	–	–	37	–	–	41	–	–	28	–	–	150
5	–	–	–	–	–	–	–	15	–	–	35	–	–	36	–	–	32	–	–	41	–	–	159
6	–	–	–	–	–	–	–	15	–	–	28	–	–	40	–	–	33	–	–	36	–	–	152
7	–	–	–	–	–	–	–	15	–	–	28	–	–	44	–	–	39	–	–	32	–	–	158
8	–	–	–	–	–	–	–	7	–	–	34	–	–	39	–	–	44	–	–	26	–	–	150
9	–	–	–	–	–	–	–	–	–	21	–	–	33	–	–	38	–	–	49	–	–	32	173
10	–	–	–	–	–	–	–	10	–	–	28	–	–	40	–	–	36	–	–	35	–	–	149
																							1525
Mortality of parental [N]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mortality of parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Immobile / Stillborn	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
juveniles
Unhatched eggs	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

– = no juveniles

M = mortality

3.1 mg/l

 

Data	12.	13.	14.	15.	16.	17.	18.	19.	20.	21.	22.	23.	24.	25.	26.	27.	28.	29.	30.	01.	02.	03.
(2002)	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	07	07	07
Replicate No.	Number of juveniles																						Total
1	–	–	–	–	–	–	–	13	–	–	23	–	–	36	–	–	40	–	–	20	–	–	132
2	–	–	–	–	–	–	–	–	–	22	–	41	–	–	61	–	–	43	–	–	–	51	218
3	–	–	–	–	–	–	–	16	–	–	27	–	–	38	–	–	41	–	–	22	–	–	144
4	–	–	–	–	–	–	–	11	–	–	32	–	–	37	–	–	37	–	–	35	–	–	152
5	–	–	–	–	–	–	–	18	–	–	36	–	–	39	–	–	31	–	–	40	–	–	164
6	–	–	–	–	–	–	–	13	–	M	–	–	–	–	–	–	–	–	–	–	–	–	–
7	–	–	–	–	–	–	–	–	14	–	29	–	–	41	–	–	35	–	–	37	–	–	156
8	–	–	–	–	–	–	–	16	–	–	33	–	–	37	–	–	32	–	–	46	–	–	164
9	–	–	–	–	–	–	–	11	–	–	20	–	–	51	–	–	42	–	–	31	–	–	155
10	–	–	–	–	–	–	–	13	–	–	33	–	–	44	–	–	40	–	–	35	–	–	165
																							1450
Mortality of parental [N]	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	0	1
Mortality of parental [%]	0	0	0	0	0	0	0	0	0	10	0	0	0	0	0	0	0	0	0	0	0	0	10
Immobile / Stillborn	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
juveniles
Unhatched eggs	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

– = no juveniles

M = mortality

6.25 mg/l

 

Data	12.	13.	14.	15.	16.	17.	18.	19.	20.	21.	22.	23.	24.	25.	26.	27.	28.	29.	30.	01.	02.	03.
(2002)	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	07	07	07
Replicate No.	Number of juveniles																						Total
1	–	–	–	–	–	–	–	–	23	–	–	35	–	–	50	–	–	38	–	–	–	50	195
2	–	–	–	–	–	–	–	–	–	24	–	–	43	–	–	51	–	–	54	–	–	18	190
3	–	–	–	–	–	–	–	16	–	–	33	–	–	39	–	–	45	–	–	32	–	–	165
4	–	–	–	–	–	–	–	17	–	–	32	–	–	36	–	–	45	–	–	39	–	–	169
5	–	–	–	–	–	–	–	9	–	–	32	–	–	38	–	–	39	–	–	36	–	–	154
6	–	–	–	–	–	–	–	14	–	–	33	–	–	35	–	–	32	–	–	38	–	–	152
7	–	–	–	–	–	–	–	14	–	–	33	–	–	30	–	–	39	–	–	41	–	–	157
8	–	–	–	–	–	–	–	–	13	–	34	–	–	27	–	–	33	–	–	43	–	–	150
9	–	–	–	–	–	–	–	10	–	–	29	–	M	–	–	–	–	–	–	–	–	–	–
10	–	–	–	–	–	–	–	9	–	–	30	–	–	41	–	–	39	–	–	37	–	–	156
																							1488
Mortality of parental [N]	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	1
Mortality of parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	10	0	0	0	0	0	0	0	0	0	10
Immobile / Stillborn	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
juveniles
Unhatched eggs	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

– = no juveniles

M = mortality

12.5 mg/l

 

Data	12.	13.	14.	15.	16.	17.	18.	19.	20.	21.	22.	23.	24.	25.	26.	27.	28.	29.	30.	01.	02.	03.
(2002)	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	07	07	07
Replicate No.	Number of juveniles																						Total
1	–	–	–	–	–	–	–	10	–	–	59	–	–	–	–	37	–	–	39	–	–	–	145
2	–	–	–	–	–	–	–	–	–	23	–	35	–	–	49	–	–	43	–	–	–	43	193
3	–	–	–	–	–	–	–	–	–	19	–	–	36	–	–	55	–	–	56	–	–	20	186
4	–	–	–	–	–	–	–	–	–	21	–	39	–	–	46	–	–	37	–	–	–	56	199
5	–	–	–	–	–	–	–	–	–	29	–	–	30	–	–	49	–	–	51	–	–	23	182
6	–	–	–	–	–	–	–	–	–	28	–	30	–	–	–	53	–	–	52	–	–	24	187
7	–	–	–	–	–	–	–	13	–	–	32	–	–	31	–	–	39	–	–	42	–	–	157
8	–	–	–	–	–	–	–	–	17	–	19	–	–	40	–	–	42	–	–	34	–	–	152
9	–	–	–	–	–	–	–	–	–	21	–	–	37	–	41	–	1	–	55	–	–	49	204
10	–	–	–	–	–	–	–	–	9	–	34	–	–	32	–	–	40	–	–	49	–	–	164
																							1779
Mortality of parental [N]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Mortality of parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Immobile / Stillborn	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
juveniles
Unhatched eggs	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

– = no juveniles

M = mortality

25 mg/l

 

Data	12.	13.	14.	15.	16.	17.	18.	19.	20.	21.	22.	23.	24.	25.	26.	27.	28.	29.	30.	01.	02.	03.
(2002)	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	07	07	07
Replicate No.	Number of juveniles																						Total
1	–	–	–	–	–	–	–	–	17	–	–	31	–	–	49	–	–	40	–	M	–	–	–
2	–	–	–	–	–	–	–	–	–	23	–	–	48	–	54	–	–	–	47	–	–	59	231
3	–	–	–	–	–	–	–	–	–	21	–	–	52	–	–	53	–	–	55	–	–	45	226
4	–	–	–	–	–	–	–	–	–	21	–	–	41	–	–	47	–	–	48	–	–	44	201
5	–	–	–	–	–	–	–	–	–	18	–	–	44	–	–	48	–	–	52	–	–	51	213
6	–	–	–	–	–	–	–	–	14	–	32	–	–	36	–	–	39	–	1	–	35	–	157
7	–	–	–	–	–	–	–	–	–	27	–	–	31	–	–	43*	–	–	56	–	–	43	200
8	–	–	–	–	–	–	–	–	12	–	–	–	31	–	19	–	27	22	–	–	50	–	161
9	–	–	–	–	–	–	–	–	–	26	–	–	31	–	–	33*	–	–	47	–	–	44	181
10	–	–	–	–	–	–	–	–	–	20	–	35	–	–	35	–	–	54	–	–	40	–	184
																							1754
Mortality of parental [N]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	1	0	0	1
Mortality of parental [%]	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	10	0	0	10
Immobile / Stillborn	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	76	0	0	0	0	0	0	76
juveniles
Unhatched eggs	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

– = no juveniles

M = mortality

* = immobilized juveniles

50 mg/l

 

Data	12.	13.	14.	15.	16.	17.	18.	19.	20.	21.	22.	23.	24.	25.	26.	27.	28.	29.	30.	01.	02.	03.
(2002)	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	06	07	07	07
Replicate No.	Number of juveniles																						Total
1	–	–	–	–	–	–	–	–	–	16	–	–	25	–	4	35	–	–	35	I	–	8	123
2	–	–	–	–	–	–	–	–	–	20	–	–	28	–	–	50	–	–	61	I	–	23	182
3	–	–	–	–	–	–	–	–	–	16	–	–	30	–	–	42	–	–	23	I	–	–	111
4	–	–	–	–	–	–	–	–	–	3	41	–	–	–	–	45	–	–	23	I	–	–	112
5	–	–	–	–	–	–	–	–	–	–	13	–	–	27	–	–	41	–	–	50 I	–	–	131
6	–	–	–	–	–	–	–	–	–	–	–	18	–	–	–	–	–	44	–	–	32	–	94
7	–	–	–	–	–	–	–	–	–	–	–	30	–	–	–	21	–	–	35	–	–	50	136
8	–	–	–	–	–	–	–	–	–	–	–	–	16	–	–	32	–	–	55	–	–	47	150
9	–	–	–	–	–	–	–	–	–	–	M	–	–	–	–	–	–	–	–	–	–	–	–
10	–	–	–	–	–	–	–	–	–	–	–	9	–	–	28	–	–	43	–	–	28	–	108
																							1147
Mortality of parental [N]	0	0	0	0	0	0	0	0	0	0	1	0	0	0	0	0	0	0	0	0	0	0	1
Mortality of parental [%]	0	0	0	0	0	0	0	0	0	0	10	0	0	0	0	0	0	0	0	0	0	0	10
Immobile / Stillborn	0	0	0	0	0	0	0	0	0	55	54	57	99	27	32	225	41	87	232	50	60	128	1147
juveniles
Unhatched eggs	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

– = no juveniles

M = mortality

I = Immobilized adult

NB:all juveniles were immobilized

Individual body length measurements

 

	Concentration (mg/l)
Vessel no.	0	3.1	6.25	12.5	25	50
1	5.2	5.1	5.1	5.2	-	4.7
2	5.1	5.4	5.8	5.3	5.9	5.3
3	5.3	5.3	5.4	5.5	6.0	5.0
4	5.3	5.3	5.4	5.5	5.5	5.0
5	5.3	5.1	5.2	5.5	5.8	4.7
6	5.4	-	5.3	5.5	5.3	5.0
7	5.4	5.1	5.3	5.3	5.6	5.0
8	5.3	4.6*	5.4	5.3	5.5	5.2
9	5.6	5.3	-	5.5	5.5	-
10	5.2	5.4	5.4	5.4	4.9	4.8

 

* = missing part of tail

M = mortality

 

Parental weight (dry)

 

Concentration	Daphnid set no.	No. of daphnids in group	Total weight (mg)	Weight per daphnid (mg)
0	1	5	6.5	1.30
0	2	5	5.9	1.18
3.1	1	5	5.9	1.18
3.1	2	4	4.9	1.23
6.25	1	5	5.7	1.14
6.25	2	4	4.6	1.15
12.5	1	5	5.1	1.02
12.5	2	5	5.8	1.16
25	1	4	4.3	1.08
25	2	5	5.2	1.04
50	1	5	2.4	0.48
50	2	4	3.4	0.85

 

Appendix VI:Statistical analysis results

 

Piperazine daphnid 21 d

File: piperazine

Transform: NO TRANSFORMATION

 

GRP	IDENTIFICATION	N	MIN	MAX	MEAN
1	0	10	140.000	173.000	152.500
2	3.1	9	132.000	218.000	161.111
3	6.25	9	150.000	195.000	165.333
4	12.5	10	145.000	204.000	176.900
5	25	9	157.000	231.000	194.889
6	50	9	94.000	182.000	127.444

 

 

Piperazine daphnid 21 d

File: piperazine

Transform: NO TRANSFORMATION

 

GRP	IDENTIFICATION	VARIANCE	SD	SEM
1	0	89.611	9.466	2.994
2	3.1	569.361	23.861	7.954
3	6.25	275.000	16.583	5.528
4	12.5	432.544	20.798	6.577
5	25	694.861	26.360	8.787
6	50	699.528	26.449	8.816

 

 

Validity criteria fulfilled:

yes

Conclusions:

21 day NOEC to Daphnia is 12.5 mg/L.

Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna STRAUS - clone 4, in a 21-day semi-static test complying with the OECD Guideline No. 211, 21st September 1998.

The test criterion of toxicity used was reproductive capacity expressed as the number of neonates per daphnid per day.

 

The nominal concentrations used in the study were as follows:

0, 3.1, 6.25, 12.5, 25 and 50 mg/L

 

Analytical determinations of the test solutions were made on six occasions during the test. The concentrations were found to remain stable to within 20% of the nominals. The nominal concentrations were used to calculate the effect concentrations.

 

The validity criteria were respected:

No mortality occurred in the control group over the test period.

The average number of juveniles per parent in the control was 152.5 after 21 days

 

Normally, the No Observed Effect Concentration (NOEC) is determined as the concentration used in the study that is immediately below the Lowest Observed Effect Concentration (LOEC), the latter derived statistically from the data using the appropriate statistical test.

 

Data was found to be normally distributed and homogeneous. Using Dunnett’s and Bonferroni-t tests, the lowest Observed Effect Concentration (LOEC) based on reproductive output, weight of adult daphnids and on parent body length was found to be 50 mg/L. Based on these statistical results the NOEC for all parameters tested is 25 mg/L.

 

However, all neonates from all broods released were immobile (but not dead) at 50 mg/l. neonates from a brood released on day 15 were observed to be immobile (but not dead) in two vessels at 25 mg/L. This concentration is therefore taken as the LOEC and the NOEC is 12.5 mg/L.

The EC50 for adult mortality and for reproduction could not be determined due to insufficient mortality in any of the test concentrations.

Reference 2

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 April 2003 - 1 May -2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

1998

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Analysis of the test solutions
For one test vessel from each test group, the test solution before and after water replacement was sampled 3 times during the exposure period, in amounts of 0.5 mL for the control, 1.0, 3.2 and 10.0 mg/L groups, and 0.2 mL for the 32.0 and 100 mg/L groups. Each sample was made up to 100 mL using purified water, and then analyzed by LC-MS. The test substance concentration of each test solution was determined from the ratio of the test solution peak area to the standard solution peak area. The details are shown in the Appended Document.

Vehicle:

no

Details on test solutions:

Prior to test solution preparation, the dilution water was aerated and kept in a thermostatic bath at 20 ± 1°C. The test substance stock solution was prepared by weighing 1000 mg of test substance and dissolve this in 1000 mL dilution water.
The test solutions were prepared by taking the respective amounts of test substance stock solution corresponding to the test concentrations shown under nominal concentrations and making the solutions up to the fixed volume of 1.0 L using dilution water. Next, for each concentration group, 80 mL test solution was delivered into each of 10 test vessels.
Dilution water with no added test substance was used as control.
At the time of preparation, the test solution (appearance) was colorless and transparent in all test groups.

Test organisms (species):

Daphnia magna

Details on test organisms:

Study organism
1) Japanese name : Oomijinko (Daphnia magna)
2) Latin name : Daphnia magna
3) Obtained from : National Institute for Environmental Studies (Environment Agency)
(Currently: National Institute for Environmental Studies (Independent Administrative Agency))
4) Date obtained : July 18, 1995
5) Sensitivity : see reference substance
6) Growth stage : Juvenile females (within 24 hours old)
7) Rearing conditions for parent Daphnia used for obtaining the test organisms:
Rearing water : Dilution water (see 3.2)
Rearing density : ≤1 organism/80 mL (25 organisms/2 L)
Rearing vessel : 2 L glass jar
Water temperatur : 20 ± 1°C
Dissolved oxygen concentration : ≥60% of saturation concentration
pH : 6.7-8.5
Illumination : Indoor light, 16 hours illumination (≤800 lux)/8 hours darkness
Rearing period : March 13, 2003 to April 10, 2003
Mortality rate of parent Daphnia in 2 weeks prior to exposure: <0%
Production of dormant eggs and males : None
Type of feed: Chlorella vulgaris (unicellular green algae) (used after the algal culture solution has been centrifuged and replaced with dilution water)
Amount of feed: 0.2 mg C (organic carbon content)/organism/day
Rearing water replacement: Replaced regularly (3 times/week). Juveniles removed daily if possible.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

±250 the detailed results are presented in attached background material

Test temperature:

19-4 - 20.6 the detailed results are presented in attached background material

pH:

7.6 - 9.8 The pH was higher in the concentration groups (7.6-9.8) than in the control group (7.6-8.3). This was attributed to the test substance.

Dissolved oxygen:

7.2 - 8.9 the detailed results are presented in attached background material

Salinity:

Elendt Medium the detailed results are presented in attached background material

Conductivity:

Elendt Medium

Nominal and measured concentrations:

Nominal concentrations (set values): Control (0 mg/L), 1.0, 3.2, 10.0, 32.0, 100 mg/L (common ratio: 3.2)
Time weighted mean measured concentrations: 1.1, 3.5, 10.0, 32.7, 100 mg/L the detailed results are presented in attached background material

Details on test conditions:

Test conditions
1) Exposure mode : Semi-static (entire volume of test solution replaced daily)
Water surface covered with Teflon sheet
2) Exposure period : 21 days
3) Test solution volume : 80 mL/vessel
4) Number of replicates : 10 vessels/test concentration
5) Number of test organisms : 10 organisms/test vessel (1 organism/vessel)
6) Test temperature : 20 ± 1°C
7) Dissolved oxygen concentration : ≥3 mg/L, preferably ≥60% saturation concentration
Test solution is not aerated during exposure period
8) pH : 6-9 (if caused by the test substance, not limited to this range) Fluctuation within 1.5
No adjustment of test solution pH
9) Hardness : Approximately 250 mg/L (calculated as CaCO3)
10) Illumination : Indoor light, 16 hours illumination (≤800 lux)/8 hours darkness
11) Feed : Type: Chlorella vulgaris (unicellular green algae) (used after the algal culture solution has been centrifuged and replaced with dilution water)
Amount of feed: 0.15 mg C (organic carbon content)/organism/day

Test procedure
The test solution temperature, dissolved oxygen concentration, pH and hardness were measured, and then the test Daphnia were introduced using a glass pipette; this moment was regarded as the start of exposure. When introducing the Daphnia, the volume of rearing water in the pipette was within 1% of the total test solution volume. Thereafter, the Daphnia were reared for 21 days; during this period, they were transferred to fresh test solution every time the water was replaced.
A fixed amount of feed was provided daily throughout the exposure period (see 3.1).

The observation of the Daphnia and the water quality measurements were performed as described below.
1) Observation of the Daphnia:
Parent Daphnia: Deaths, motility status and the presence/absence of abnormal appearance were observed daily and recorded. Any dead Daphnia were removed.
Juveniles born: The number of surviving juveniles was counted and recorded daily from the first brood production.
The presence/absence of dead juveniles, aborted eggs and dormant egg production were observed and recorded. The day the first brood was produced (day of first brood production) was recorded.
2) Water quality measurements: The water temperature, dissolved oxygen concentration, pH and hardness were measured for one test vessel per test group, before and after water replacement, 4 times during the exposure period.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

21 d

Dose descriptor:

LC50

Remarks:

Parental mortality

Effect conc.:

57.5 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: 95% CI: 32.7 - 101 mg/L

Duration:

21 d

Dose descriptor:

EC50

Remarks:

Ìnhibition of reproduction

Effect conc.:

66.2 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

32.7 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

101 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

Tables presenting the detailed results are presented in attached background material

Results with reference substance (positive control):

Concentration of standard substance (potassium dichromate, special reagent grade) affording 50% immobilization after 48 hours (EiC50) = 0.60 mg/L (95% confidence interval: 0.52-0.69 mg/L).
This value is almost the sameas the EiC50 value at this institute (from June 1998, n = 9).
Mean value ± standard deviation = 0.77 ± 0.18 mg/L
Minimum value to maximum value = 0.57 to 1.02 mg/L

Reported statistics and error estimates:

Maximum no observed effect concentration (NOEC) and lowest observed effect concentration (LOEC)*
The cumulative number of juveniles per surviving parent for the 21-day period was calculated for each test vessel, the presence or absence of a significant difference between each concentration group and the control group was determined by statistical methods**, and the maximum no observed effect concentration (NOEC) and the lowest observed effect concentration (LOEC) were determined.

*Maximum no observed effect concentration (NOEC): The highest concentration at which no significant reproductive inhibition is observed compared to the control Lowest observed effect concentration (LOEC): The lowest concentration at which significant
reproductive inhibition is observed compared to the control
**Statistical methods: Bartlett's test of homogeneity of variances, one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison test

The Yukms software, Statlight “#4 comparison of multiple groups” (Yukms Corp, Tokyo) was used for the statistical analysis.

Environmental factors thought to have influenced the reliability of the study results: There were no applicable events.

Test substance concentration in the test solution

The test substance concentration in the test solution before and after water replacement was measured 3 times during the exposure period. The results are shown in Table 1.

Analysis of the test solutions (see 3.6) revealed that some measured values exceeded ±20% of the set value, and so the time-weighted mean of the measured values was used when calculating the results described below (50% lethal concentration in parent Daphnia, 50% reproduction inhibition concentration, maximum no observed effect concentration and lowest observed effect concentration).

Daphnia observation results

Parent Daphnia deaths and mortality rate

The cumulative number of dead parent Daphnia and the mortality rate in each test group, for the exposure period, are shown in Table 2-1, Table 2-2 and Figure 1.

The mortality rate of the parent Daphnia in the control group was 10% on completion of exposure, which satisfied the study validation criteria of ≤20%. In the highest concentration

group, the mortality rate was 100% on completion of exposure.

Day of first brood production

The day of first brood production for the parent Daphnia in each test group are shown in Table 3.

The day of first brood production for the parent Daphnia in the control group was within 9 days of the start of exposure, which was judged to be within the normal range. In the highest concentration group, all parent Daphnia died before first brood production.

Mean cumulative number of juveniles

The mean cumulative number of juveniles per parent Daphnia in each test group, during the exposure period, are shown in Table 4 and Figure 2. The mean cumulative number of juveniles per parent Daphnia in the control group for the 21-day period was 102 juveniles, which satisfied the study validation criteria of ≥60 juveniles.

In the highest concentration group, all parent Daphnia died before first brood production.

Production of dormant eggs etc.

Production of dormant eggs was not observed in any test group, throughout the exposure period.

50% Lethal concentration (LC50) in parent Daphnia

The 50% lethal concentration (LC50) in parent Daphnia on 21-day exposure is shown in Table 5 below.

21-day LC50: 57.5 mg/L (95% confidence interval: 32.7-101 mg/L)

50% Reproduction inhibitory concentration (EC50)

The 50% reproduction inhibitory concentration (EC50) on 21-day exposure is shown in Table 6 below.

21-day EC50: 66.2 mg/L (95% confidence interval: could not be calculated)

Table 5: Calculated LC50 values for parental daphnia

Exposure period	LC50*	95% confidence intervals	Statistical method
(day)	(mg/L)	(mg/L)
21	57.5	32.7 - 101	Binomial

*Based on time weighted mean measured concentrations

Table 6: Calculated EC50 values for inhibition of Reproduction

Exposure period	EC50*	95% confidence intervals	Statistical method
(day)	(mg/L)	(mg/L)
21	66.2	-	Logit

Ba *Based on time weighted mean measured concentrationssed on time

Maximum no observed effect concentration (NOEC) and lowest observed effect

concentration (LOEC) on cumulative number of juveniles

The maximum no observed effect concentration (NOEC) and the lowest observed effect

concentration (LOEC) on the cumulative number of juveniles per parent Daphnia on 21-day

exposure are shown in Table 7 and below.

21-day NOEC: 32.7 mg/L

21-day LOEC: 101 mg/L

Test solution temperature, dissolved oxygen concentration, pH and hardness

The test solution temperature, dissolved oxygen concentration, pH and hardness throughout the exposure period are shown in Table 8, Table 9, Table 10 and Table 11, respectively.

The water temperature was 20 ± 1°C in all test groups, and the dissolved oxygen concentration was ≥60% of the saturated oxygen concentration (saturated oxygen concentration at 20°C:

8.8 mg/L) in all test solution tanks, and so both satisfied the study criteria. The pH was higher in the concentration groups (7.6-9.8) than in the control group (7.6-8.3). This was attributed to the

test substance. The hardness was judged to be within appropriate range (around 250 mg/L).

Detailed results and description of the test medium used are presented in the attached background material.

Validity criteria fulfilled:

yes

Remarks:

The validity criteria were respected: Mortality in the control group did not exceed 20% (1 mortality on day 20) over the test period. The average number of juveniles per parent in the control was 101.7 after 21 days.

Conclusions:

The 21 day NOEC for reproduction of daphnia magna due to exposure to piperazine is 32.7 mg/L using the time weighted average measured concentration.

Executive summary:

The purpose of this study was to assess the toxicity of the test substance dissolved in fresh water, on the reproductive efficacy of Daphnia magna in a 21-day semi-static test complying with the OECD Guideline No. 211, 21st September 1998.

The test criterion of toxicity used was reproductive capacity expressed as the number of neonates per daphnid per day.

 

The nominal concentrations used in the study which were based on the results observed in the acute daphnia test (48h EC50 = 106 mg/L) were as follows:

Control (0 mg/L), 1.0, 3.2, 10.0, 32.0, 100 mg/L (common ratio: 3.2)

 

Analytical determinations of the test solutions were made during the test. Most of concentrations were found to remain stable to within 20% of the nominals but one concentration deviated more than 20% and therefore the time weighted average concentrations were used to calculate the effect concentrations.

 

The validity criteria were respected:

Mortality in the control group did not exceed 20% (1 mortality on day 20) over the test period.

The average number of juveniles per parent in the control was 101.7 after 21 days

 

The lowest Observed Effect Concentration (LOEC) based on reproductive output was found to be 101 mg/L. Based on these statistical results the NOEC for reproduction is 32.7 mg/L.

 

The LC50 for adult mortality is 57.5 mg/L (95% CI:.32.7 - 101 mg/L)

Description of key information

There are two long term toxicity tests with daphnia magna available. The exposure concentrations were monitored in both studies and in both studies the test substance was observed to be stable during the test and therefore the nominal test concentration was used for the calculation of the endpoints.

The study from Thomas (2002) resulted in a NOEC of 12.5 mg/L based on the immobility of Neonates and a NOEC of 50 mg/L for reproduction

The study from Mitsubishi (2003) resulted in a NOEC of 32.7 mg/L for reproduction.

Both studies are considered of equal quality. 21d NOEC of 12.5 mg/L will be used in the dossier as a worst-case.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Remarks:

Immobility of Neonates; NOEC reproduction = 50 mg/L

Effect concentration:

12.5 mg/L