Lupine, L. albus, ext.

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:

19 September 2016 to 27 September 2016

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:

yes

Remarks:

For the concentration 10 mg/L in the abiotic conditions (test item only, without algae), the deviation between T0 and T72 hours is higher than 20% unlike the stability test performed. The deviation was still higher than 20 % with additional analysis.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

14 January 2016

Analytical monitoring:

yes

Details on sampling:

Chemical analyses of the test item were performed in the test solutions (biotic and abiotic). In order to verify the nominal or initial concentrations of the test item and maintenance of the exposure concentrations of the test item during the ecotoxicological testing, chemical analysis of the test item in algae test solutions were performed in samples containing the test item from algae ecotoxicology solutions by a Total Organic Carbon analyzer and the following protocol:

- at the beginning of the test (T = 0 h)
- at the end of the test (T = 72 h)
- for the control (i.e. algae test medium filtered), the highest and the lowest concentrations and a concentration around expected EC50 (filtered solutions)

A total of 8 analyses were performed at the start of the test (T 0 h).
A total of 8 analyses were performed at the end of the test (T 72 h).

Schedule analysis at initial time (T0h):
Control Abiotic: 10 mg/l Abiotic; 328 mg/L Abiotic; 1050 mg/L Abiotic.
Schedule analysis at final time (T72h):
Control Biotic: 10 mg/l Biotic; 328 mg/L Biotic; 1050 mg/L Biotic.
Control Abiotic: 10 mg/l Abiotic; 328 mg/L Abiotic; 1050 mg/L Abiotic.

The measured concentrations of the test item should not vary more than ± 20 % of nominal or initial measured concentration during the test.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
The test item is soluble in the test medium.
The test medium and the test solutions were filtered through a membrane filter (cellulose acetate) with a mesh size of 0.22 µm to sterilizing and all the manipulations have been done under sterile conditions to prevent contamination.
Five test item concentrations in algae test medium (OECD medium) were performed, plus a control.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Strain no. CCAP 278.4
- Method of cultivation: Algae cultures are stored in the laboratory on a solid medium (LC/agar Petri plates) and in a liquid medium with regular sub-culturing (every week).

Test type:

not specified

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

No data

Test temperature:

22.6–23.4 °C shaking at 120 rpm

pH:

pH at the start: 7.2 - 8.0
pH after 72 h: 7.6 - 7.8

Dissolved oxygen:

No data

Salinity:

No data

Conductivity:

No data

Nominal and measured concentrations:

10, 32, 102, 328 and 1050 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Initial cells density: 10000 cells/mL at the start of the test
- No. of vessels per concentration (replicates): 3 biotic replicates + 1 abiotic replicate per concentration
- Control: 6 biotic replicates + 2 abiotic replicates

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Algae test medium (OECD medium); Algae dilution water

OTHER TEST CONDITIONS
- Light intensity and quality: 6378 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The algae growth inhibition in relation to the control was evaluated over a period of 72 h. The algae cells per unit volume were measured during the test at 24, 48 and 72 h with a particle counter (Beckman Coulter Z2) and under a microscope with a Malassez counting cell.
- pH: pH of each test concentration was measured at the start and at the end of the test. The pH of the control medium had not increased by more than 1.5 units during the test.

TEST CONCENTRATIONS
- Range finding study: 10, 100 and 200 mg/L
- Results used to determine the conditions for the definitive study: Algae concentrations (x 10^4 per mL of test solution) i.e., 222.1, 160.3, 69.3 and 25.7 at 0, 10, 100 and 200 mg/L, respectively at 72 h. Based on the results of the preliminary test, the following test concentrations 10, 32, 102, 328 and 1050 mg/L were proposed for the definitive test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 10 - < 32 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.:

> 1 050 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 10 - < 1 050 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

See "any other information on results incl. tables"

Results with reference substance (positive control):

The last algae growth inhibition test performed with the reference item potassium dichromate (August 23, 2016) gave an ErC50 (0-72h) = 0.75 mg/L (0.66 - 0.85), indicating that the algae sensitivity is correct (within the accepted interval of 0.65 to 1.73 mg/L fixed by the International Standard NF EN ISO 8692 from May 2012 as mentioned in the OECD Guideline) and in accordance with previous data obtained by the laboratory.

Table 6.1.5/1: Algae growth inhibition

Nominal test item concentration		[algae] *10^4/mL at 24 hours			[algae] *10^4/mL at 48 hours			[algae] *10^4/mL at 72 hours
		Count.	Average	cv	Count.	Average	cv	Count.	Average	cv
Control	a	6,1	6,2	4,9	30,3	31,9	5,5	177,8	171,6	3.5
	b	6,2			32,9			179,3
	c	6,4			31,0			166,3
	d	5,8			34,9			165,0
	e	5,9			30,5			168,4
	f	6,6			32,0			172,6
10 mg/L	a	6,8	6,6	5,7	32,3	29,5	11,8	152,5	149,5	5.9
	b	6,2			25,6			139,5
	c	6,9			30,5			156,4
32 mg/L	a	6,0	6,2	3,3	30,5	31,6	3,2	84,2	83,8	5.1
	b	6,4			31,7			79,4
	c	6,3			32,5			87,9
102 mg/L	a	6,3	6,2	3,3	32,8	33,6	5,1	81,5	87,2	5.6
	b	6,4			35,6			90,2
	c	6,0			32,5			89,8
328 mg/L	a	6,1	6,0	5,1	24,3	27,5	15,1	88,0	93,6	5.4
	b	6,3			32,2			97,8
	c	5,7			26,0			95,1
1050 mg/L	a	3,5	3,5	5,7	22,8	21,7	5,6	42,2	45,8	7.2
	b	3,7			20,4			48,7
	c	3,3			21,9			46,5

 

CV = Coefficient of variation.

 

Table 6.1.5/2: Specific growth rate

 

Control replicates		Specific growth rate
		0-24h	Average	CV (%)	24 -48h	Average	CV (%)	48 -72h	Average	CV (%)	0 - 72h	Average	CV per replicate (%)	Average CV
Control	a	1.808	1.818	2.7	1.603	1.644	5.0	1.770	1.682	4.2	1.727	1.715	6.3	6.7%
	b	1.825			1.669			1.696			1.730		4.8
	c	1.856			1.578			1.680			1.705		8.3
	d	1.758			1.795			1.553			1.702		7.6
	e	1.775			1.643			1.709			1.709		3.9
	f	1.887			1.579			1.685			1.717		9.1

CV: Coefficient of variation (%)

Table 6.1.5/3: Calculation of the E_rC₅₀(0-72h)

Nominal test item concentration		Algae *10^4/mL at 0hour	Average growth rate µ0-72h	Inhibition growth rate (% Ir)	Average inhibition of growth rate (%)	Coefficient of variation	Significance
Control	a	1	1,727	Not applicable		0.7	-
	b		1,730
	c		1,705
	d		1,702
	e		1,709
	f		1,717
10 mg/L	a	1	1,676	2,3	2,7	Not required	*
	b		1,646	4,0
	c		1,684	1,8
32 mg/L	a	1	1,478	13,8	14,0	Not required	*
	b		1,458	15,0
	c		1,492	13,2
102 mg/L	a	1	1,467	14,5	13,2	Not required	*
	b		1,501	12,5
	c		1,499	12,6
328 mg/L	a	1	1,492	13,0	11,8	Not required	*
	b		1,528	10,9
	c		1,518	11,5
1 050 mg/L	a	1	1,247	27,3	25,7	Not required	*
	b		1,295	24,5
	c		1,280	25,4

The average specific growth rate (µ) was determined according to the OECD guideline.

Statistical significance was determined with the Dunnett test (α=5%) from raw data with the scientific software Minitab 17.2.1. Therefore, it was not possible to determine a NOEC because a significant difference between the control was observed at the lowest concentration 10 mg/L.

The results did not permit to calculate a regression because the inhibition of growth rate at 1050 mg/L is only 25.7%.

The raw data did not permit to determined ErC10-(0-72h) with reliability, for information the ErC10-(0-72h could be  between 10 and 32 mg/L.

The results obtained for the test item on the toxicological endpoint “inhibition of growth rate” indicate an ErC50-(0-72h) higher than 1050 mg/L (highest tested concentration)  value expressed in nominal test item concentration

Calculation of the EyC₅₀(0-72h)

The results did not permit to calculate a regression because the Yield inhibition were too close between 32 and 328 mg/L.

The results obtained for the test item on the toxicological endpoint “Yield inhibition” indicate anEyC₁₀-(0-72h) is inferior 10 mg/L(lowest tested concentration), value expressed in nominal test item concentration.

The results obtained for the test item on the toxicological endpoint “Yield inhibition” indicate anEyC₅₀-(0-72h)betwwen 10 and 1050 mg/L,value expressed in nominal test item concentration.

 

Table 6.1.5/3: Results of TOC analysis measured in test solutions

 

Nominal test item concentration	mg “test item”/L in algae solutions		Variation between T0 and T72 hrs (%)
	Results at T0 hour(deviation/nominal value)	Results at T72 hours
Control	< 5	48.8	NA
10 mg/L	8.4 (+ 16.0%)	55.5	+ 560
328 mg/L	315 (+ 4.1%)	315	0
1050 mg/L	1011 (+ 3.7%)	950	6

TOC = Total Organic Carbon

Under test conditions (with algae), the test item is considered to be stable for the nominal concentrations 328 and 1050 mg/L, except 10 mg/L, because the measured concentrations of the test item did not vary by more than ± 20% of the initial concentrations during the test. At 10 mg/L and in Control, the increase of Total Carbon during the testing was explained by the growth of algae due to lack of inhibition effect at 10 mg/L.

Validity criteria

In the control, the increase of the biomass during 72 h was determined to be higher than a factor of 16.

The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35 % and 7% in the control.  

The validity criteria were successfully fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 72 h ErC10, ErC50, EyC10 and EyC50 of the test item was determined to be between 10 and 32 mg/L, > 1050 mg/L, < 10 mg/L and between 10 and 1050 mg/L, respectively.

Executive summary:

Introduction

In an algal growth inhibition study performed according to OECD Guideline 201 and in compliance with GLP, freshwater green algae species Pseudokirchneriella subcapitata was exposed to test item at 10, 32, 102, 328 and 1050 mg/L over an exposure period of 72 h. The algae growth inhibition in relation to the control was evaluated over a period of 72 h. The algae cells per unit volume were measured during the test at 24, 48 and 72 h with a particle counter (Beckman Coulter Z2) and under a microscope with a Malassez counting cell.

Chemical analysis

In order to verify the nominal or initial concentrations of the test item and maintenance of the concentratrations during the ecotoxicological testing, TOC analysis were performed in test solutions. Under test conditions (with algae), the test item is considered to be stable for the nominal concentrations 328 and 1050 mg/L because the measured concentrations of the test item did not vary by more than ± 20% of the initial concentrations during the test. At 10 mg/L and in Control, the increase of Total Carbon during the test was explained by the growth of algae due to lack of inhibition effect.

Results

A concentration effect relationship was observed and statistically analyzed to obtain effect concentrations.Under the test conditions, the 72 h ErC10, ErC50, EyC10 and EyC50 of the test item was determined to be between 10 and 32 mg/L, > 1050 mg/L, < 10 and between 10 and 1050 mg/L, respectively.

Validity criteria

Regarding the validity criteria of the study, the increase of the biomass during 72 h was determined to be higher than a factor of 16 in the Control. The coefficients of variation of daily section-by-section specific growth rate and of average specific growth rate were determined respectively to be less than 35 % and 7% in the control. Therefore, the validity criteria were fulfilled.

 

Description of key information

Under the test conditions, the 72 h ErC50 of the test item to the algae Pseudokirchneriella subcapitata was determined to be > 1050 mg/L.

Under the test conditions, the 72 h ErC10 of the test item to the algae Pseudokirchneriella subcapitata was determined to be between 10 and 32 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

1 050 mg/L

EC10 or NOEC for freshwater algae:

10 mg/L

Additional information

An algal growth inhibition study was available and it was performed according to OECD Guideline 201 and in compliance with GLP. In this study, the freshwater green algae species Pseudokirchneriella subcapitata was exposed to the test item at 10, 32, 102, 328 and 1050 mg/L over an exposure period of 72 h. The algae growth inhibition in relation to the control was evaluated over a period of 72 h.

The validity criteria were fulfilled and a concentration effect relationship was observed and statistically analyzed to obtain effect concentrations.

Under the test conditions, the 72 h ErC10, ErC50, EyC10 and EyC50 of the test item was determined to be between 10 and 32 mg/L, > 1050 mg/L, < 10 and between 10 and 1050 mg/L, respectively.