AERO 5100 PROMOTER

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:

09 - 11 Nov 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No NOEC could be derived since test concentrations were not selected appropriately.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and solvent control
- Sampling method: Duplicate samples of 200 mL were taken from solvent control and test cultures at 0 h and at 72 h (replicates pooled) for analysis. The samples were not filtered to remove algal cells prior to analysis. Additional samples were also taken from flasks containing the test substance at a concentration equivalent to the highest test concentration but with no algae present, at 72 h (replicates pooled), in order to obtain information on the extent of adsorption/absorption of the test substance by the algal cells.

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An aliquot (2.2 g) of test substance was dissolved in 10 mL of dimethylformamide to give an initial stock solution of 220 mg/mL. This solution was diluted with dimethylformamide to prepare the stock solution series: 220, 100, 46, 22, 10 and 4.6 mg/mL.
120 µL of the appropriate stock solution or solvent only was spiked into 1200 mL screw top bottles filled to capacity with algal medium to exclude air and minimise losses of test substance. Each bottle was placed into an oscillating incubator overnight to aid dispersion. After stirring, each bottle was inoculated with algal cells through a septum to produce a culture with a cell density of approximately 1.0 x 10^4 cells/mL. 200 mL of these test and control cultures were then decanted into the exposure vessels, 600 mL capacity stoppered bottles.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 100 µL/L

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: CCAP 278/4
- Source: Algal cultures were obtained from the Culture Collection of Algae & Protozoa, Institute of Freshwater Ecology, Cumbria, UK.
- Pre-culture: Sterile nutrient medium was inoculated from a master culture and cultured under continuous illumination (ca. 7000 lux) in an orbital incubator at 23 ± 2 °C, to give an algal suspension in log phase growth, characterised by a cell density of 2.8 x 10E+05 cells/mL

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

24 ± 1 °C

pH:

7.6 - 8.1 (start of exposure)
8.5 - 9.6 (end of exposure)

Nominal and measured concentrations:

control, solvent control, 0.46, 1.0, 2.2, 4.6, 10, 22 mg/L (nominal)
control, < LOQ, 0.33, 0.74, 1.6, 3.4, 7.2, 16 mg/L (mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed (stoppered bottles)
- Material, size, headspace, fill volume: 600 mL vessels (Screw top bottles) filled with 200 mL solution/cultures, leaving 400 mL headspace
- Aeration: Gaseous exchange and suspension of the algal cells were ensured by the action of the orbital shaker, oscillating at 120 cycles per minute
- Initial cells density: 9.7 x 10E+03 cells/mL (control), 1.0 x 10E+04 cells/mL (solvent control)
- Control end cells density: 4.5 x 10E+05 cells/mL (control), 4.8 x 10E+05 cells/mL (solvent control)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 6 (100 µL auxiliary solvent per litre)

GROWTH MEDIUM
- Standard medium used: yes (according to OECD 201 with a slightly higher amount of 80 mg/L of FeCl3.6H2O)

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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: 7 x 30 W "universal white" 1 metre fluorescent tubes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell densities of the control and test cultures at 0, 24, 48 & 72 h were determined by direct counting using a particle counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approx. 2.2
- Range finding study: yes
- Test concentrations: 0.1 to 10 mg/L

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 8.3 - 10 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.33 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: There was statistically significant inhibition of growth (at the 5% level) at this concentration, however the mean % inhibition was <10% which is not considered biologically significant

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CI: 3.4 - 4.4 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none

Reported statistics and error estimates:

For percentage inhibition, Williams' test (Williams, 1971, 1972) was used to compare each treated group with the baseline (control) values and Bartlett's test for homogeneity of variance (Bartlett, 1937) was also applied. The data were entered by hand and analysed using SAS 6.11 (SAS Institute 1989).

BARTLETT, M.S. (1937) Properties of sufficiency and statistical tests. Proceedings of the Royal Society. Series A, 160, 268-282.
SAS INSTITUTE (1989) SAS/STAT User's Guide, Version 6, Fourth Edition, Vol.2. SAS Institute Inc., Cary, NC, USA.
WILLIAMS, D.A. (1971) A test for differences between means when several dose levels are compared with a zero dose control. Biometrics, 27, 103-117.
WILLIAMS, D.A. (1972) The comparison of several dose levels with a zero dose control. Biometrics, 28, 519-531.

Analytical measurements and test design:

The mean measured concentrations at 0 hours indicate that test substance was lost during the preparation of the exposure concentrations. This was due to the high volatility of the test substance (100% volatile by weight). Various attempts were made to conduct the study with the exclusion of air from the test vessels to reduce these losses. This resulted in direct inhibition of growth to an unacceptable level, with concurrent stimulation of bacterial growth.

The test vessels used in the definitive study were sealed bottles containing test medium and allowing a 400 mL head space. These conditions promoted normal algal growth rates and reduced bacterial growth. The mean measured concentrations at 72 hours were all between 68 and 77% of their respective nominal concentrations (74 to 80% of nominal at 0 hours), indicating that when in solution, the test substance formed a stable dispersion. Comparison of the measured concentrations of the test substance in samples at a concentration equivalent to the highest test concentration, but without the addition of algal cells, indicated that the presence of algal cells had not affected the stability of the exposure solutions.

Table 1: Inhibition of growth

Nominal concentration [mg/L]	Mean measured concentration[mg/L]	Area under curve @ 72 hours	% Inhibition*	Growth rate (0-72 hours)	% Inhibition*
Control	-	-	-	-	-
Solvent control	ND	14	0	0.053	0
0.46	0.33	13	9.3	0.052	3.4
1.0	0.74	12	11	0.047	11
2.2	1.6	11	21	0.048	11
4.6	3.4	7.7	45	0.044	18
10	7.2	4.6	68	0.032	41
22	16	1.2	92	0.017	69

* Percentage inhibition values calculated using non-rounded data

ND: None detected

Table 2: Measured concentrations - Mean values and percentages of nominal

Nominal concentration [mg/L]	Number of samples analysed	Mean* measured concentration [mg/L]	% Nominal
Solvent control	3	Solvent control	-
0.46	3	0.33	72
1.0	3	0.74	74
2.2	j	1.6	73
4.6	3	3.4	73
10	3	7.2	72
22	3	16	73

* Arithmetic mean of 0 and 72 hour measured concentrations

Table 3: Summary of analytical chemistry results

Occasion	Nominal conc. [mg/L]	Measured concentration [mg/L]		Mean measured concentration [mg/L]	Recovery as % Nominal
		Analysis 1	Analysis 2
0 hours	Solvent Control	ND	ND	ND	-
	0.46	0.3527	0.3496	0.3512	76.3
	1.0	0.7889	0.8114	0.8002	80.0
	2.2	1.679	1.743	1.711	77.8
	4.6	3.402	3.459	3.431	74.6
	10	7.372	7.571	7.472	74.7
	22	16.17	-	16.17	73.5
72 hours	Solvent Control	ND	-	ND	-
	0.46	0.3136	-	0.3136	68.2
	1.0	0.6880	-	0.6880	68.8
	2.2	1.519	-	1.519	69.1
	4.6	3.295	-	3.295	71,6
	10	6.988	-	6.988	69.9
	22	15.90	-	15.90	72.3
	22 (no algae)	16.90	-	16.90	76.8

ND: None detected

Table 4: Summary of analytical chemistry results - procedural recoveries

Occasion	Fortified concentration [mg/L]	Recovery as a % of fortified concentration
0 hours	Control	-
	2.2698	92.5
	2.2698	93.7
0 hours - analysis of duplicates	Control	-
	2.2698	98.7*
	2.2698	98.6
72 hours	Control	-
	2.2698	100
	2.2698	101
	25.22**	90.7
	25.22**	91.2
Mean (RSD)		95.8 (4.4)

ND: None detected

* Mean of two analyses

** Additional recoveries performed due to change in test levels

Description of key information

ErC50 (72 h): 9.3 mg/L (mean measured concentration, OECD 201)

NOErC (72 h): < 0.33 mg/L (mean measured concentration, OECD 201)

Additional information

One experimental study is available investigating the effects of the substance to aquatic algae (Cytec Industries, 1998). The study was performed according to OECD 201 (GLP) using Pseudokirchneriella subcapitata as test organism. A stock solution of the substance in dimethylformamide was prepared to obtain the final test concentrations of 0.46, 1.0, 2.2, 4.6, 10 and 22 mg/L. In addition a test medium control and solvent control were tested. The actual test concentrations were measured by GC/FID analysis resulting in mean measured concentrations near nominal concentrations (0.33, 0.74, 1.6, 3.4, 7.2, 16 mg/L). During exposure an inhibition of growth was recorded resulting in an ErC50 (72 h) of 9.3 mg/L (95% CI: 8.3 - 10 mg/L) and a NOErC (72 h) of < 0.33 mg/L based on mean measured concentrations.