1,4-Diazabicyclo[2.2.2]octane-2-methanol

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:

03 - 06 Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Water samples were taken from each test group at 0 and 72 hours for quantitative analysis.
- Sampling method: At the end of the exposure, equal amount of the test solution was taken out from all replicates in each test group and mixed. The mixture was centrifuged for 10 min at 3000 rpm and the supernatant was used for analysis.

Vehicle:

no

Details on test solutions:

- Method: 0.1 g of test substance (adapted to purity) was dissolved in test medium to prepare 1000 mg/L stock solution. Three test vessels (300 mL glass Erlenmeyer flask with air-permeable silicone rubber stopper) were prepared for each test concentration group. The stock solution and the test medium of the requirement were taken into the test vessel to prepare 100 mL of test solution. Six test vessels containing 100 mL of test medium were prepared in the control group. For measurement of environmental conditions (temperature and pH), one additional vessel was prepared in all test groups.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Unicellular green algae
- Strain: ATCC22662
- Source (laboratory, culture collection): Summit Pharmaceuticals International Corporation, ATTC Division

ACCLIMATION
- Acclimation period: Preculture was carried out under the same conditions as in the test (period from 27 Feb - 3 Mar 2014)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 ± 2 °C (22.2 - 23.4 °C)

pH:

start: 8.29 - 9.03
end: 7.78 - 8.19

Nominal and measured concentrations:

Nominal: 0, 9.5, 17, 31, 56 and 100 mg/L
Measured: not detected, 4.56, 17.2, 30.0, 48.1, 89.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL glass Erlenmeyer flask with air-permeable silicone rubber stoppers
- Fill volume: 100 mL/vessel
- Aeration: constantly shaken (100 rpm)
- Initial cells density: 5E+03 cells/mL
- Control end cells density: 6.38E+03 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (sterilized OECD medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to OECD 201

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity: mean light intensity during exposure 5366 – 6006 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: cell density of each flask was determined using a Coulter counter.
- Other: Cell abnormalities were recorded microscopically.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
Range finding study:
- Test concentrations: nominal test concentrations of 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Inhibition values after 72 h were 1.93% and 46.0%, respectively.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

9.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.56 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 89.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Unusual cell shape: No abnormalities were observed in the control and test vessels.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: ErC50 (0-72 h): 1.2 mg/L (95% CL: 0.76 – 1.8 mg/L)
- The standard substance potassium dichromate was tested in December 2013.

Reported statistics and error estimates:

- Regarding the growth rate, Bartlett's test was conducted to determine the absence of homogeneity of variance
- The NOEC value was determined by Kruskal-Wallis rank sum test and Dunnett's nonparametric multiple comparison test

Within the exposure period, the biomass in the control increased by a factor of 128 times (validity criterion ≥ 16 times).

The mean coefficient of variation for growth rates in the control was 27% (validity criterion ≤ 35%).

The coefficient of variation of average specific growth rates in replicate controls was 0.25% (validity criterion ≤ 7%).

 

Table 1: Inhibition of Growth Rate

Nominal concentration (Measured concentration) [mg/L]		Growth Rate
		0-72 h	% inhibition
Control	R1	1.6194	-
	R2	1.6173
	R3	1.6107
	R4	1.6182
	R5	1.6131
	R6	1.6216
	Mean	1.6167
9.5 (4.56)	R1	1.6185	-0.111
	R2	1.5791	2.33
	R3	1.6141	0.161
	Mean	1.6039	0.793
17 (17.2)	R1	1.5843	2.00
	R2	1.5877	1.79
	R3	1.5838	2.04
	Mean	1.5853	1.94*
31 (30.0)	R1	1.5517	4.02
	R2	1.5471	4.31
	R3	1.5427	4.58
	Mean	1.5472	4.30*
56 (48.1)	R1	1.3384	17.2
	R2	1.3238	18.1
	R3	1.3343	17.5
	Mean	1.3322	17.6*
100 (89.6)	R1	1.0789	33.3
	R2	1.0589	34.5
	R3	1.0425	35.5
	Mean	1.0601	34.4*

R = Replicate

* = Significantly different compared to the control (α = 0.05)

Table 2: Summary of analytical results

Nominal concentration [mg/L]	Analyzed concentration [mg/L]			Percentage residue
	0 h	72 h	Mean measured concentration
9.5	10.4	< 2.0	4.56	< 19
17	20.1	14.8	17.2	74
31	33.0	27.3	30.0	83
56	51.7	44.7	48.1	86
100	94.1	85.4	89.6	91

 

 

Validity criteria fulfilled:

yes

Description of key information

EC50 (72 h): > 100 mg/L (nominal) / > 89.6 mg/L (measured) for Pseudokirchneriella subcapitata (OECD 201)
NOEC (72 h): 9.5 mg/L (nominal) / 4.56 mg/L (measured) for Pseudokirchneriella subcapitata (OECD 201)

Additional information

One experimental study investigating the effects of test substance to aquatic algae is available (Tosoh Corporation, 2014). The study was performed according to OECD 201 under GLP conditions with the freshwater algae Pseudokirchneriella subcapitata. A test with five test concentrations of 9.5, 17, 31, 56 and 100 mg/L (nominal) was conducted under static test conditions. At the highest test concentration growth inhibition of 34.4% was observed, whereas at the test concentration of 9.5 mg/L no significant inhibition of growth was observed. Thus, an EC50 (72 h) > 100 mg/L (nominal), corresponding to > 89.6 mg/L (measured), and a NOEC (72 h) = 9.5 mg/L (nominal), corresponding to 4.56 mg/L (measured), based on both growth rate were derived.