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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 - 18 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Name: Piperazine
Synonym: Hexahydropyrazine, diethylenediamine
CAS no.: 110-85-0
Mol formula: C4H10N2
Molecular weight: 86.14 Dalton
Boiling point: 146 °C
Melting Point: 111.5 °C
Vapour pressure: 21 Pa at 20 °C
Solubility: Soluble in alcohol, glycerin, glycol, insoluble in ether
Water solubility: ≥ 1000 mg/L
Specific Gravity: 1.1
Log Kow: -1.17
Analytical monitoring:
yes
Details on sampling:
The test was performed semi-static renewal of the test solution every 24 hours.
The test concentration was monitored at the start and end of this time interval
Vehicle:
no
Details on test solutions:
The state (appearance) of the test solution at the time of preparation was colorless and transparent in all test groups.
Test organisms (species):
Oryzias latipes
Details on test organisms:
1) Japanese name : Himedaka
2) Scientific name : Oryzias latipes
3) Source : Inhouse culture
4) Egg collection date : 13 November 2002
5) Auxiliary number : 02-H-1113B
6) Scale length : average length: 1.77 cm (1.65 – 1.95 cm), n = 10
7) Weight : average weight: 0.068 g (0.050 – 0.095 g), n = 10
8) Sensitivity : 96 hours LC50 with reference substance (copper sulfate (H) pentahydrate, reagent special grade) Degree [LC50) = 0.69 mg /L (95% confidence interval: 0.50-0.95 mg/L). This value is in good agreement with the historical LC50 value at our institute (since 1995, n = 24).
a. Mean ± standard deviation = 0.93 ± 0.29 mg/L.
b. Minimum value to maximum value = 0.44 to 1.5 mg/L
9) Adaptation period : March 24, 2003-April 14, 2003 2 Mortality on child days prior to exposure was less than 5%, macroscopically healthy and normal. Individuals were used. (The conditions for swelling for 14 days before the start of exposure are shown below.
10) Adaptation conditions
a. Breeding water: Dilution water (see 3.2)
b. Breeding method: Running water type (breeding density: 1.0 g/L/ day or less)
c. Temperature : 24 ± 1 °C
d. Dissolved oxygen concentration: 80% or more of saturation concentration
e. pH: 6.5 – 8.5
f. Light conditions : Room light, 16 hours light (1000 lux or less) / 8 hours dark
g. Feeding amount: Approximately 2% of fish body weight / day (no feeding from 24 hours before the start of exposure)
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
73 mg/L total hardness
Test temperature:
24 ± 0.7 °C
pH:
7.8 - 9.8
Dissolved oxygen:
6.9 - 8.5
Conductivity:
170 µS/cm
Nominal and measured concentrations:
Nominal: Control: 0 mg/L Limit test concentration: 100 mg/L (nominal)
Measured: Control: 0 mg/L Limit test concentration: 102 mg/L (nominal)
Details on test conditions:
Test conditions:
1) Exposure method: Semi-static (replace the entire amount of test solution every 24 hours), cover the water surface with a Teflon sheet
2) Exposure period : 96 hours
3) Test solution volume : 5 Liter/container. The weight of the test fish per 1.0 L of the test solution was not exceeded 1.0 g
4) Number of replicates : 1 container / test vessel
5) Number of test organisms : 10 fish/ test vessel
6) Test temperature : 24 ±1 °C
7) Dissolved oxygen concentration: maintained at 60% or more of saturation concentration (no aeration)
8) The pH of the test solution was not adjusted.
9) Light conditions : Room light, 16 hours bright (1000 lux or less) / 8 hours dark
10) Feeding : No feeding
11) Fish monitoring frequency : Lethatlity and sub-lethal effect were monitored at least every 24 hours.
12) pH measurements: : Every refreshment old and new (table 8)
13) Temperature measurements: Every refreshment old and new (table 6)
14) Dissolved Oxygen monitoring frequency: very refreshment old and new (table 7)
Reference substance (positive control):
yes
Remarks:
copper sulfate pentahydrate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
Geomean measured concentration during the 24h interval was 102 mg test item/L or 101.4 mg active ingredient/L
Basis for effect:
mortality (fish)
Details on results:
The limit test was performed based on the results of the preliminary range finding test which was performed with 5 fish per test concentration.
Results with reference substance (positive control):
96 hours LC50 with reference substance (copper sulfate (H) pentahydrate, reagent special grade) LC50 observed = 0.69 mg/L (95% confidence interval: 0.50-0.95 mg/L).
This value is in good agreement with the historical LC50 value at our institute (since 1995, n = 24).
a. Mean ± standard deviation = 0.93 ± 0.29 mg/L.
b. Minimum value to maximum value = 0.44 to 1.5 mg/L
Reported statistics and error estimates:
The test is performed as a limit test at 100 mg/L and no lethality and also no other effects were observed compared to the control.
Sublethal observations / clinical signs:

Table 1: Measured concentration of the test substance in test water:

Nominal concentration       Measured concentration (mg/L)       Geometric mean measured concentration (mg/L)

0 hours (new)       24 hours (old)

Control (0 mg/L)                     <0.3                    <0.3                                   ----

100 mg/L                                   106                    98.8                                   102

Table 2: Mortality of the Medaka (Oryzias lapites) exposed to the test substance

Nominal concentration       Geomean measured concentration       Cumulative Mortality (percent Mortality)

(mg/L)                                   (mg/L)                                   24 hours       48 hours       72 hours       96 hours

Control                                          0                                          0 (0)              0 (0)              0 (0)        0 (0)

100                                           102                                         0 (0)              0 (0)             0 (0)          1 (10)    

As a result of the analysis of the test solution, the ratio of the measured values to the nominal concentration was all within ± 20%. The nominal concentration was therefore used to calculate the result.

Table 3: Calculated LC50 values: 24, 48, 72 and 96 hours > 100 mg/L

Table 4: Observation of the highest concentration in 0% mortality and the lowest concentration in 100% mortility:

24, 48, 72 and 96 hours > 100 mg/L

Table 5: Observed Toxicological Symptomes: No toxicological symptomes were observed after 24, 48, 72 and 96 hours

Table 6 Dissolved Oxygen Concentrations:

Nominal                                   Dissolved Oxygen Concentration (mg/L)

Concentration       0 Hour              24 Hour              48 Hour              72 Hour       96 Hour

(mg/L)                     new            old        new       old        new       old       new       old

Control                     8.4        7.4       8.4        7.0         8.4       7.2         8.3       6.9     

100                           8.5            7.3       8.5        7.1         8.5       7.0         8.4       6.9

Table 8 pH values:

Control pH range: 7.5 - 7.8 measured at start and at each refreshment in old and new solutions

100 mg/L pH range: 9.7 - 9.8 measured at start and at each refreshment in old and new solutions

Validity criteria fulfilled:
yes
Remarks:
1) mortality did not exceed 10% in the control; 2) the dissolved Oxygen concentration in the test vessels was >60%; 3) Analytical measurement of the test concentrations was performed;
Conclusions:
Reliable and valid study
a) the test procedure is reported (e.g. composition of the test medium (see attached picture);
b) The dissolved oxygen, pH, and temperature measured prior to and after each water renewal are reported (see results). The results of hardness and TOC determinations in the dilution water are reported (see attached picture);
c) adequate information on the analytical method (including performance parameters of the method) and on the results of the analytical determination of exposure concentrations are provided;
d) mortalities and sub-lethal effects (e.g. with regard to equilibrium, appearance, ventilator and swimming behaviour) are reported. The fish are inspected at least after 24, 48, 72 and 96 hours
Executive summary:

The acute toxicity of Piperazine (CAS no 110 -85 -0) was tested according to OECD 203 (1992) under GLP conditions. The test was performed as a limit test at 100 mg/L test item based on the results of the range finding test. The test is performed semi-statically with 24 h refreshment of the test solutions. The exposure concentrations during the test were confirmed by specific chemical analysis using LC/MS. These analytical measurements showed that Piperazine was stable under test conditions and therefore the nominal test concentration was used for the calculation of the endpoint.

During the 96 h of exposure to 100 mg/L piperazine no lethality and also no other sub-lethal effects were observed leading to the conclusion that the 96h LC50 fish > 100 mg a.i./L

Description of key information

The short-term toxicity to fish has been tested using three different fish species. Tests with two freshwater species Oryzias latipes and Poecilia reticulata and one with the marine species Cyprinodon variegatus.

The exposure concentrations during the test were however only monitored in the study with Oryzias latipes (Mitsubishi, 2003). This test is therefore considered as 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 96h LC50 of piperazine for fish is observed to be > 100 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Dose descriptor:
LC50
Effect concentration:
> 100 mg/L

Additional information