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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 - 11 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
no
Principles of method if other than guideline:
The following is adviced by the guideline (1984) in relation to: Replicates and controls
The test design should include preferably three replicates at each test concentration and ideally twice that number of controls. In this study three controls were used. This is not expected to reduce the reliability of this test because the standard deviation of the observed cell densities in the controls is very low and cell densities of the lowest test concentration (30 mg/L) are identical. The NOEC observed = 110 mg/L
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At the start of exposure (0 hr), 2.0 mL of the test solution was collected from each of the three test vessels in each test group and mixed, and the sample was used as an analysis sample. At the end of exposure (72 hr), 2.0 mL of the test solution was collected from each of the three test containers in each test group and mixed, and the supernatant after centrifugation (3000 rpm, 10 minutes) of algae was used as an analysis sample.

for the control group and the concentration group less than 70.0 mg/L, 100 μL of each analysis sample was diluted to 100 mL with purified water and then analyzed by LC/MS. For concentration groups more than 110 mg/L, 50 μL of each analytical sample was diluted to 200 mL with purified water and then analyzed by LC/MS. The test substance concentration of each test solution was quantified according to the ratio of the peak area of the standard solution. Details are shown in Appendix-2.
Vehicle:
no
Details on test solutions:
Preparation of test solution.
The medium used to prepare the test solution was 23 ± 2 °C in a constant temperature bath or constant temperature room before preparation.  
The test substance stock solution was prepared as shown in the table below.

Test substance stock solution
Amount of test substance collected 500 mg
Dissolving aid None
Constant capacity
(Constant solution: OECD medium) 500 mL
Test substance concentration 1000 mg/L
Auxiliary agent concentration --

100 mL test medium was put in each test container and the amount of medium which test substance solution will be added was removed. Each test solution was prepared by adding it in a geometric progression. The control solution was medium only.
The state (appearance) of the test solution at the time of preparation was colorless and transparent in all test groups.

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
Test organism
1) Japanese name : Muremikazukimo (unicellular green algae)
2) Scientific name : Selenastrum capricornutum
3) Strain name : ATCC22662
4) Source : American Type Culture Collection
5) Date of acquisition : 20 June 1996
6) Management after acquisition : Aseptic subculture using Gorham medium
7) Preculture: : Pre-culture period: April 4, 2003-April 8, 2003
During this time, the algae grew logarithmically. (Environmental conditions are the same as the test)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Remarks on exposure duration:
According to guideline
Post exposure observation period:
Microscopical observation: no change in cell morphology (contraction, swelling / rupture, etc.) or cell aggregation was observed in all concentration groups, and there was no difference observed from the control group.
Hardness:
OECD standard test medium was used
Test temperature:
Temperature:
Exposure period Temperature (°C)
0 22.8
24 23.0
48 22.7
72 22.9
pH:
Nominal concentration (mg/L) pH at T0 pH at T72
Control 7.8 8.9
30 9.4 8.9
46 9.6 8.8
70 9.7 8.8
110 10.0 8.7
160* 10.1 8.6/8.4/8.4
250 10.2 8.6
* pH in each vessel was measured because the cell densities were significantly different

Piperazine is a base which is like other amine containing substances (e.g. ammonia) expected to have a higher toxicity at higher pH values.
Considering the observed cell densities during the first 24 hours this pH effect is expected to be minimal.
Dissolved oxygen:
100 RPM shaking
Salinity:
OECD standard test medium was used
Conductivity:
OECD standard test medium was used
Nominal and measured concentrations:
Nominal test concentrations: Control group, 30.0, 46.0, 70.0, 110, 160, 250 mg/L (ratio 1.5)
Observed test concentrations T0: <0.3, 31.7, 42.3, 63.5, 92.3, 155 and 207 mg/L
Observed test concentration T72: <0.3, 14.4, 27.7, 59.9, 108, 150 and 238 mg/L
The main cause of the observed decrease at the lower test concentrations was thought to be adsorption to algal cells.
Details on test conditions:
Test method
Test conditions:
1) Exposure method: Water-stop type, shaking culture (100 rpm)
2) Exposure period: 72 hours
3) Test solution volume: 100 mL (OECD medium according to OECD TG 201, see below)/ test vessel
4) Number of replicates: 3 test vessels/test concentration
5) Initial cell concentration: Pre-cultured algae 1x10^4 cells/mL
6) Test temperature: 23 ± 2 ℃
7) Lighting: Continuous illumination at 4000 Lux (within ± 20% fluctuation, near flask liquid level)

medium
  For both preculture and testing, the medium specified in the OECD Chemicals Test Guidelines was used.
 The composition table is shown below

Test vessels and algae culture test equipment, etc.
1} Test vessel : 300 mL glass Erlenmeyer flask (with breathable silicon stopper)
2) Algae culture test equipment :Ito Seisakusho AGP-150RL
3) Optical microscope 2 : Nikon ECLIPSE TE300
4) Particle counting device : Sysmex CDA-500
5) Electrolytic solution for particle counting device: Sysmex Cell Back
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

Test operation
The number of cells of the pre-cultured algae was counted, and a certain amount of the pre-cultured solution was added to the container containing the test solution so that the cell concentration in the test solution was 1x10^4 cells/mL.
Each test vessel was placed in an incubator at 23 ± 2 °C. to start the test, and the cell concentration was measured 24, 48 and 72 hours later. For cell concentration, 1.0 mL of test solution (0.5 mL for 72 hr) was collected from each test container. After mixing with 9.0 mL (9.5 mL at 72 hr) of the electrolytic solution (cell pack) for the particle counter, the measurement was performed with the particle counter (CDA-500).

Test medium used: Standard OECD test medium
NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 0.08 mg/L
Na2EDTA.2H20 0.1 mg/L
H3BO3 0.185 mg/L
MnCl2.4H2O 0.415 mg/L
ZnCl2 0.003 mg/L
CoCl2.6H2O 0.0015 mg/L
CuCl2.2H2O 0.00001 mg/L
Na2MoO4.2H2O 0.007 mg/L
NaHCO3 50 mg/L
The pH of this medium after equilibration with air is approximately 8.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
153.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
a.i. is 99.4%
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
109.3 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
a.i. is 99.4%
Basis for effect:
growth rate
Details on results:
EC values were calculated according to OECD TG 201.
Results with reference substance (positive control):
Confirmation of sensitivity : 72 hours 50% growth with reference substance (potassium dichromate, special grade reagent)Inhibition concentration (EbC50) = 0.318 mg/L [95% confidence interval = 0.207 to 0.489 mg/L). This value in good agreement with the historical EbC50 value at our institute shown below (since November 1996, n = 11) Mean ± standard deviation = 0.443 ± 0.0751 mg/L  

Reported statistics and error estimates:
EC values were calculated according to OECD TG 201.
Linear regression analysis (least squares method) was performed using points where linearity is observed on the concentration lifetime growth inhibition rate curve and calculate from the intersection with the inhibition rate of 50%.


Maximum inactive concentration (NOEC)
After confirming homoscedasticity by performing Bartlett's homoscedastic test (α = 0.01), perform one-way analysis of variance (1-way ANOVA, α = 0.05) and Williams' multiple comparison test (α = 0.05, both sides). The highest test concentration for which there is no statistical significant difference of effect when compared to the control group was defined as the maximum no observed concentration (NOEC). At that time, NOECb (0-72) was used when calculated by the area method, and NOECr (24-48) or NOECr (24-72) was used when calculated by the velocity method. When the concentration groups of 160 and 250 mg / L were included, homoscedasticity could not be confirmed, NOECr (24-72) was used. For excluded concentration groups, after confirming homoscedasticity by performing F-test (α = 0.05) with the control group, the significant difference is confirmed according to Student's t-test (α = 0.05, both sides) or Welch's t-test (α = 0.05, both sides)
For the above statistical analysis, Yukms software Statlight "# 4 multi-group comparison" or "# 3 2 group comparison" (Yukms Corp, Tokyo) was used.

Minimum value to maximum value = 0.285 to 0.543 mg/L

Results and considerations

Preliminary test results 

First time

Second time

Concentration (mg/L)

Growth rate after 72 hours relative to the control(%)

Concentration (mg/L)

Growth rate after 72 hours relative to the control (%)

Controlarea

100

Controlarea

100

1.00

84

50.0

121

10.0

81

110

12

100

27

230

1

1000

0

500

0

 

Environmental factors that seemed to affect the reliability of test results

There were no relevant events.

Concentration of test substance in test solution

The concentration of the test substance in the test solution was measured at the start of exposure (0hr) and at the end of exposure (72hr). The results are shown under "nominal and measured concentrations".

As a result of the analysis of the test solution, for keeping the ratio of the nominal test concentration compared to the measured concentration within ± 20% at the start of exposure, the nominal test concentration is used to derive the dose-response.

The test substance concentration at 72 hours of exposure was 14.4 to 238 mg/L, and the ratio to the set value was 48 to 98%. The decrease was slightly larger at lower concentrations. The main cause of the observed decrease in concentration was thought to be the adsorption to algal cells.

5.3 Growth curve

The cell concentration in the control group increased by an average of 175 times after culturing for 72 hours, showing normal growth under the test conditions. The cell concentration in each concentration group tended to decrease (dose-dependently) as the concentration increased.

In addition, in the observation 48 hours after the start of exposure, the test solution was macroscopically greenish in the control group and the concentration group less than 110 mg/L. After that, the greenness tended to increase with the passage of time. In the observation 72 hours after the start of exposure, the test solution was macroscopically greenish in the 160 mg/L concentration group. The 250 mg/L concentration group did not turn green until 72 hours. As a result of observing the cell morphology under a microscope at the end of the exposure, no change in cell morphology (contraction, swelling / rupture, etc.) or cell aggregation was observed in all concentration groups, and there was no difference from the control group.

5.4 50% growth inhibition concentration (EC50) and maximum no observed effect concentration (NOEC)

Table 3 shows the growth inhibition rate in the concentration group, Table 4 shows the 50% growth inhibition concentration (EC50) and the maximum no observed effect concentration (NOEC), and Figure 2 and Figure 3 show the concentration-inhibition rate curve. From the above results, the following conclusions were obtained.

1) Inhibition concentration by comparison of the area under the growth curve

EbC50 (0-72): 91.2 mg/L (95% confidence interval: 58.3 to 143 mg/L)

NOECb (0-72): 46.0 mg/L

2) Inhibition concentration by comparison of growth rate

ErC50 (24-48): 140 mg/L (95% confidence interval: cannot be calculated)

NOECr (24-48): 70.0 mg/L

ErC50 (24-72): 154 mg/L (95% confidence interval: cannot be calculated)

NOECr (24-72): 110 mg/L

5.5 Temperature and pH

Table 5 shows the temperature inside the culture test equipment during the 72-hour exposure period, and Table 6 shows the pH of the test solution.

The temperature inside the culture test device was within the set range (23 ± 2 °C). The pH of the test group was 7.8 to 10.2 at the start of exposure, as pH related to concentration was high, which is is judged to be caused by the test substance. It was 8.4 to 8.9 at the end of the exposure. If carbon dioxide assimilation is active and the growth rate of algae is high, the pH will increase by more than 1. This time, the pH increased by more than 1 in the control group.

Validity criteria fulfilled:
yes
Remarks:
validity criteria – The cell concentration in the control cultures increased by a factor 175 within three days. The pH of the control solutions increased 1.1 where they normally deviate not more than one unit during the test.
Executive summary:

The algae growth inhibition by Piperazine (CAS no 110 -85 -0) was tested according to OECD 201 (1984) under GLP conditions. The test was performed with nominal test concentrations of 0 (control), 30.0, 46.0, 70.0, 110, 160 and 250 mg/L (ratio 1.5)

and was based on the results of range finding tests. 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 only in the sub-NOEC test vessels lower recoveries lesss than 80% after 72h exposure were observed. This decrease is most likely caused by sorption of the positively charged test substance to algae. The nominal test concentration was therefore used for the calculation of the endpoints. The 72 h NOEC was 109.3 mg a.i./L and the 72 h ErC50 was 153.1 mg a.i./L.

Observed test concentrations T0: <0.3, 31.7, 42.3, 63.5, 92.3, 155 and 207 mg/L

Observed test concentration T72: <0.3, 14.4, 27.7, 59.9, 108, 150 and 238 mg/L

The main cause of the observed decrease at the lower test concentrations was thought to be adsorption to algal cells.

Description of key information

There are three toxicity tests with aquatic algae available. Two tests with the freshwater species Pseudokirchneriella subcapitata and one with the marine species Skeletonema costatum.

The exposure concentrations during the test were however only monitored in one study with Pseudokirchneriella (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 72h ErC50 of piperazine for algae was observed to be 153.1 mg/L.

The 72h ErC10 of piperazine for algae was observed to be 109.3 mg/L

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
1 000 mg/L

Additional information