1,4-dimethylpiperazine

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

Study period:

2013-11-25 - 2014-01-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Identification: Dimethylpiperazine
- Description: Pale yellow liquid
- Batch: 1903-3/2012
- Purity: 99.82%
- Expiry date: 31 December 2014
- Storage conditions: Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the control (replicates R1 - R6 pooled) and each test group (replicates R1 - R3 pooled) at 0 and 72 hours for quantitative analysis.
- Sampling method: Samples were taken at 0, 24, 48 and 72 hours and the cell densities determined using a Coulter® Multisizer Particle Counter.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

- 100 mg/L stock solution (50 mg in 500 mL culture medium)
- Lower test concentrations prepared by serial dilution using the primary stock solution
- The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute,
Oban, Argyll, Scotland
- Age of inoculum (at test initiation): not stated

ACCLIMATION
- Acclimation period: none
- Culturing media and conditions (same as test or not): The culture medium used for both the range-finding and definitive tests was the same as that
used to maintain the stock culture
- To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

none

Hardness:

not reported

Test temperature:

24 +/- 1 °C

pH:

Definitive test:
Start: 7.5
End: 7.8

It was noted during the range-finding test that the pH of the test preparations at 0 hours increased from pH 7.4 at 0.10 mg/L through to pH 9.3 at 100 mg/L. After discussion with the Sponsor, and following the recommendations of the test guideline, it was considered appropriate to adjust the pH of the definitive test preparations prior to the addition of algal cells at 0 hours to pH 7.5 in order to overcome any possible effects of the alkaline test conditions.

Dissolved oxygen:

not reported

Salinity:

not applicable

Nominal and measured concentrations:

Range finder:
0.10, 1.0, 10 and 100 mg/L
Definitive:
Nominal concentrations: control, 1.0, 3.2, 10, 32, 100 mg/L
Determined concentrations:
at 0 hrs: < LOQ, 1.0, 3.23, 9.77, 31.3, 99.7 mg test item/L
at 72 hrs: < LOQ, 0.993, 8.4*, 10.0, 30.7, 100 mg test item/L


* original sample gave a higher than expected result outside the calibration range, therefore the frozen duplicate sample was analyzed.
% of nominal concentration range was 96 - 101% except for the higher value of 8.4 mg/L (% of nominal concentration was 263).

Details on test conditions:

TEST SYSTEM
- Test vessel: glass conical flask
- Test vessels covered or not: closed with polyurethane foam bungs and incubated (INFORS Multitron, Version 2 incubator)
- Material, size, headspace, fill volume: glass, 250 mL, 100 mL fill volume
- Aeration: only by nominal orbital shaking at 100-150 rpm
- Initial cells density: 5x 10^3
- Control end cells density: 10^4-10^5 cells/mL. Cell concentration of the control cultures increased by a factor of 145 after 72 hours
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- One blank vessel (without algal inoculum) was incubated for each test concentration and the culture medium control (for background corrections)
- Random position in incubator, changed daily
- samples were taken from the control and each test group for quantitative analysis at 0 hours (from the bulk preparation) and at 72 hours (from the replicated pooled). All 0-hour samples were stored frozen prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test. The shape and size of the algal celss was inspected microscopically and any abnormalities recorded.

GROWTH MEDIUM
- Standard medium used: yes (AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: with 1M HCl to appropriate range
- Photoperiod: 24 h light
- Light intensity and quality: warm white lighting (380 – 730 nm), 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): algal cell density (calculated parameters: biomass and growth rate) was determined after 24, 48 and 72 h
- Determination of cell concentrations: Coulter electronic particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1-3.3

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- Effect concentrations exceeding solubility of substance in test medium: none
- Inhition of yield:
EC10: > 100 mg/L
EC20 > 100 mg/L
EC50> 100 mg/L
- Validation criteria: the increase in the cell control cultures increased by a factor of 145 after 72 hours. The mean coefficient of variation for growth rate fort eh control cultures was 6%. The coefficient of variation for averate specific groth rate for the controle cultures was 1%

Results with reference substance (positive control):

reference substance: 

ErC50 (0 – 72 h) : 1.2 mg/L; 95% confidence limits 1.0 – 1.3 mg/L

EyC50 (0 – 72 h) : 0.52 mg/L; 95% confidence limits 0.45 – 0.62 mg/L

No Observed Effect Concentration (NOEC) based on growth rate: 0.25 mg/L

No Observed Effect Concentration (NOEC) based on yield: 0.25 mg/L

Lowest Observed Effect Concentration (LOEC) based on growth rate: 0.50 mg/L

Lowest Observed Effect Concentration (LOEC) based on yield: 0.50 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

NOEC and LOEC values were determined by one-way analysis of variance incorporation Bartlett's test for momgeneity of variance (Sokal and Rolf, 1981) and Dunnett's procedure to identify significant differences (p<0.05) between individual treatments and the culture medium control.

Table 1: Daily specific growth rates for the control cultures in the definitive test

	Daily specific growth rate (cells/ML/hour)
	day 0-1	day 1-2	day 2-3
R1	0.067	0.07	0.073
R2	0.071	0.075	0.070
R3	0.072	0.076	0.066
R4	0.076	0.073	0.071
R5	0.068	0.077	0.066
R6	0.073	0.073	0.063
Mean	0.071	0.074	0.068

The mean coefficient of variation of section by section specific growth for the control cultures was 6%

Table 2: Inhibition of growth rate and yield in the definitive test

nominal concentration (mg/L)	pH 0h	cell densities * (cells per mL) 0h	cell densities * (cells per mL) 24h	cell densities * (cells per mL) 48h	cell densities * (cells per mL) 72h	pH 72h
Control R1 R2 R3 R4 R5 R6 Mean	7.5	5.33E+03 5.70E+03 6.00E+03 6.54E+03 5.7E+03 5.44E+03 5.78E+03	2.53E+04 2.73E+04 2.84E+04 3.06E+04 2.56E+04 2.84E+04 2.76E+04	1.37E+05 1.64E+05 1.77E+05 1.75E+05 1.64E+05 1.66E+05 1.64E+05	7.91E+05 8.78E+05 8.69E+05 9.56E+.5 7.93E+05 7.49E+05 8.39E+05	7.8
1.0 R1 R2 R3 Mean	7.5	5.87E+03 6.66E+03 6.51E+.3 6.35E+03	2.44E+04 2.63E+04 2.56E+04 2.54E+04	1.57E+05 1.72E+05 1.53E+05 1.61E+05	7.96E+05 8.94E+05 6.95E+05 7.95E+05	7.8
3.2 R1 R2 R3 Mean	7.5	5.66E+03 5.52E+03 5.28E+03 5.49E+03	3.03E+04 3.02E+04 2.48E+04 2.84E+04	1.61E+05 1.52E+05 1.63E+05 1.59E+05	7.2E+05 7.71E+05 8.09E+05 7.67E+05	7.8
10 R1 R2 R3 Mean	7.5	6.05E+03 6.06E+03 5.58E+03 5.9E+03	2.71E+04 2.67E+04 2.24E+04 2.54E+04	1.6E+05 1.51E+05 1.15E+05 1.42E+05	9.10E+05 8.2E+05 6.19E+05 7.83E+05	7.8
32 R1 R2 R3  Mean	7.5	6.00E+03 5.86E+03 5.74E+03 5.87E+03	2.87E+04 2.26E+04 2.51E+04 2.55E+04	1.71E+05 1.32E+05 1.57E+05 1.53E+05	8.66E+05 6.84E+05 8.53E+05 8.01E+05	7.8
100 R1 R2 R3 Mean	7.5	6.34E+03 5.92E+03 5.98E+03 6.08E+03	2.5E+04 2.84E+04 2.79E+04 2.71E+04	1.55E+05 1.57E+05 1.65E+05 1.59E+05	8.14E+05 6.67E+05 7.67E+05 7.49E+05	7.8

* cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

The coefficient of variation for average specific growth rate for the control cultures over the test period was 1%.

R1-R6 = replicates 1to 6

 

Results for test samples

Time point (hours)	nominal concentration of test item in test sample c nom (mg/L)	measured concentration of test item in sample vial mg/L	sample preparation factor (F)	determined concentration of test item in test sample (mg/L)	% of nominal concentration
0	Control 1.0 3.2 10 32 100	<LOQ 1.00 0.979 0.977 0.948 0.997	1 1 3.3 10 33 100	< LOQ 1.00 3.23 9.77 31.3 99.7	- 100 101 98 98 100
72	Control 1.0 3.2 10 32 100	< LOQ 0.993 0.632 1.0 0.93 1	1.0 1.0 13.3 10 33 100	<LOQ 0.993 8.4* 10.0 30.7 100	- 99 263* 100 96 100

LOQ = limit of quantification

- = Not applicable

* = original sample gave a higher then expected result outside the calibration range, therefore the frozen duplicate sample was diluted and analysed.

 

Validity criteria fulfilled:

yes

Conclusions:

Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal with the exception of the 3.2 mg/L test sample at 72 hours. Initial analysis showed the response to be outside of the linear range whilst analysis of the duplicate sample showed a measured concentration of 8.4 mg/L was obtained. Given that a measured concentration of 101% of nominal was observed at 0 hours it was considered that the test solution had been correctly prepared and that possible post study contamination had occurred. As such, given that the test concentration was below the No Observed Effect Concentration it was considered appropriate to base the results on the nominal test concentrations only. Exposure of Pseudokirchneriella subcapitata to the test item gave a 72-hr EC50 value > 100 mg/L. The 72-hr NOEC was 100 mg/L.

Description of key information

The information for toxicity to aquatic algae and cyanobacteria is currently being updated according to the ECHA Final Decision CCH-D-2114571346-45-01/F (deadline 11-01-2024).

A full study was performed under GLP conditions and according to OECD TG 201 (Huntsman, 2014). The 72-h EC50 for the test substance based on growth rate was > 100 mg/L. The NOEC was 100 mg/L. As a worst-case, a value of 72 -h EC50 = 100 mg/L was used for the PNEC derivation.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

In a 72-hour static test with the green alga Pseudokirchnerella subcapitata according to the OED 201 guideline, the toxicity of the test substance to algae was investigated (Vrijenhoef, 2014). Tested concentrations were: 3.2, 10, 32, 100 mg/L. After 72 hours an EC50 of > 100 mg/L for the test substance was estimated for the growth rate, related to the nominal concentration. The 72-h NOEC was 100 mg/L.