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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:

2015-09-20 to 2015-09-24

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Water accommodated fraction
In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose the organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Using this approach, aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase.

Preparation of test solutions
For the single water accommodated fraction (WAF) 100 mg/L of the test item were weighed out and transferred into a brown glass flask with dilution water one day prior to application. The WAF was shaken with 20 rpm for 24 hours at room temperature. After a separation phase of at least 1 hour the WAF was taken from the homogeneous liquid phase from the bottom of the water body.

Test loading
Per definition of the WAF all terms related to concentration level are given as loading level because partly dissolved compounds and mixtures cannot be related to concentrations. The water accomodated fraction (WAF) will be tested in a limit test.

Test concentrations
One limit concentration of 100 mg/L was tested. The limit concentration is based on the results of a preliminary range finding test (non GLP).

Control
Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: Pseudokirchneriella subcapitata HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG)
Pflanzenphysiologisches Institut der Universität Göttingen
Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A three day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE • m-2 • s-1 for 24 hours per day.

ACCLIMATION
- Acclimation period: three days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

min: 22:0
max: 23.0
mean: 22.5

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
100 8.01 9.07
Control 8.10 8.95

Nominal and measured concentrations:

nominal: 100 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 250 mL, with headspace,100 mL
- Initial cells density: 6153 cells/ml
- Control end cells density: 2666336 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 0

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 hours/day cool daylight
- Light intensity and quality: 60 - 120 µE • m-2 • s-1 , within ± 15 % over incubation area


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Type and frequency of measurement
Cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No Self-fluorescence was found in the range finding test.

Microscope evaluation
Microscopic evaluation of the cells at the start and end of the exposure was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

Reference substance (positive control):

yes

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:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Microscopic evaluation of the cells at the start and the end of the exposure period revealed no morphological abnormalities. All effect values are given based on the WAF prepared with a nominal loading concentration of 100 mg/L.

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA, batch number MKBQ9179V, purity 100.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2015-04-14 to 2015-04-17 (with headspace). The reference item toxicity is in the valid range following test facility SOPs.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.613 0.810 ± 0.404
95 % confidence interval 0.589 - 0.638
Yield inhibition
EyC50 0.281 0.421 ± 0.274
95 % confidence interval 0.234 - 0.314
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

NOEC and LOEC values
NOEC/LOEC were determined by calculation of statistical significance of growth rate and yield. As standard a t-test was used for NOEC/LOEC calculations. When running a t-test a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

Software
The data for the tables in this report were computer generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.

Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
100	1	6153	42932	445275	2339481
	2	6153	37714	437497	2376862
	3	6153	44911	489963	2621345
	4	6153	45867	449351	2356917
	5	6153	41333	424389	2649820
	6	6153	42164	444773	2517794
	Mean	6153	42487	448541	2477037
Control	1	6153	45090	500594	2925913
	2	6153	41963	431758	2489758
	3	6153	39401	438626	2596571
	4	6153	44324	480617	2575622
	5	6153	41097	455937	2647687
	6	6153	47586	489900	2762465
	Mean	6153	43244	466239	2666336

 

 

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-).

 

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
100	1		1.98	2		2333328	12
	2		1.99	2		2370709	11
	3		2.02	0		2615192	2
	4		1.98	2		2350764	12
	5		2.02	0		2643667	1
	6		2.01	1		2511641	6
	Mean	(+)*	2.00	1	(-)	2470884	7
Control	1		2.06			2919760
	2		2.00			2483605
	3		2.02			2590418
	4		2.01			2569469
	5		2.02			2641534
	6		2.04			2756312
	Mean		2.02			2660183

* = biologically not significant

 

Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

 

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.99	2.41	1.77	2.06	0.325	15.8	16.2
	2	1.92	2.33	1.75	2.00	0.298	14.9
	3	1.86	2.41	1.78	2.02	0.344	17.1
	4	1.98	2.38	1.68	2.01	0.354	17.6
	5	1.90	2.41	1.76	2.02	0.341	16.8
	6	2.05	2.33	1.73	2.04	0.301	14.8
				Mean	2.02
				SD ±	0.02
				CV [%]	0.95

CV= Coefficient of variation

SD= Standard deviation

Validity criteria fulfilled:

yes

Conclusions:

In this study the test item was found not to inhibit the growth rate of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively.
All effect levels are given based on the water accommodated fraction prepared with a nominal concentration of 100 mg/L of Lthe test item.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 (2011 )and Council Regulation (EC) No.761/2009/Method C.3 (2008) at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2015-09-20 to 2015-09-24 with the definitive exposure phase from 2015-09-21 to 2015-09-24. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6153 cells/mL. A water accommodated fraction with a nominal concentration of 100 mg/L was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

No analytical determination was carried out since no suitable method for the determination of the test item could be established. All effect values of LICOWAX R21 S FL are given based on the nominal concentration.

 

NOEC, LOEC, EC - values of the test item (0-72 hours)

based on the water accommodated fraction, prepared with 100 mg/L

                               

	Growth Rate Inhibition Nominal test item concentration [mg/L]
NOEC	100
LOEC	> 100
ErC10	> 100
ErC20	> 100
ErC50	> 100
	Inhibition of Yield Nominal test item concentration [mg/L]
NOEC	100
LOEC	> 100
EyC10	> 100
EyC20	> 100
EyC50	> 100

Description of key information

In this study the test item was found not to inhibit the growth rate of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours. The NOEC-value of the test item for both inhibition of growth rate and inhibition of yield after 72 hours was 100 mg/L. The EC50-values of the test item for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were both > 100 mg/L, respectively.

All effect levels are given based on the water accommodated fraction prepared with a nominal concentration of 100 mg/L of Lthe test item.

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

100 mg/L