Enzymatic hydrolysis products of Ophiopogon japonicus, Liliaceae, root

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:

08 February to 07 March 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:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Remarks:

14 January 2016

Analytical monitoring:

yes

Details on sampling:

Chemical analyses of “OF14 AT” were performed in biotic and abiotic replicates of test solutions at the beginning and after 72 h.
- at the beginning of the test (T = 0 h)
- at the end of the test (T = 72 h)
- for the filtered control (i.e. algae test medium filtered), the highest and the lowest concentrations and a concentration around expected EC50
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 each time
Filtered control Biotic and Filtered control Abiotic: 10, 102 and 1050 mg/L
The measured concentrations of the “OF14 AT” 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
- Method: Test item is soluble in water. Five test solutions were prepared in the test medium by weighing the test item and by direct dissolution in test medium. Then, test solutions were filtered by using a filter size of 0.22 µm in order to avoid contamination by microorganisms and thus biodegradation under experimental conditions.

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:

22.6–23.2 °C shaking at 120 rpm

pH:

pH at the start: 7.8-7.9
pH after 72 h: 6.8-9.0

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 + 2 abiotic replicates per concentration
- No. of vessels per control (replicates): 6
- Filtered 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: 6508 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 for the concentrations 328 and 1050 mg/L at 72 h in order to distinguish test substance particles from Pseudokirchneriella subcapitata alga cells.
- 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: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Algae concentrations (x 10^4 per mL of test solution) i.e., 273.7, 252.6, 212.1 and 144.7 at 0, 1, 10 and 100 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.:

33.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 16.6-102.7

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

245.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % CI: 125.6-426.5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: lowest tested concentration

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

67.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95 % CI: 41.7-106.8

Results with reference substance (positive control):

The last algae growth inhibition test performed with the reference item potassium dichromate (September 07, 2015) gave an ErC50 (0-72h) = 0.85 mg/L, 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	4.3	3.7	9.6	25.9	22.7	10.1	186.0	183.9	3.2
	b	3.9			20.9			179.7
	c	3.8			24.4			187.3
	d	3.6			24.7			183.2
	e	4.1			22.5			182.6
	f	3.9			24.6			175.6
Filtered control	a	3.2			19.8			177.8
	b	3.1			18.5			180.0
	c	3.6			21.1			194.7
	d	3.6			22.1			194.0
	e	3.5			22.7			180.5
	f	3.4			25.0			185.8
10 mg/L	a	4.2	4.0	8.1	25.0	22.5	10.0	151.9	157.0	6.2
	b	4.1			21.9			168.2
	c	3.6			20.6			150.9
32 mg/L	a	3.1	3.4	12.1	22.2	20.4	8.6	138.8	131.8	4.6
	b	3.9			18.7			127.6
	c	3.3			20.4			129.0
102 mg/L	a	2.3	2.3	2.5	10.2	11.5	9.8	74.9	76.3	5.2
	b	2.4			12.2			73.2
	c	2.3			12.1			80.8
328 mg/L	a	1.5	1.5	10.0	5.7	5.7	1.0	5.8	5.1	14.8
	b	1.7			5.8			4.3
	c	1.4			5.7			5.2
1050 mg/L	a	1.0	1.0	6.0	5.6	5.4	3.9	1.8	1.5	20.8
	b	1.0			5.2			1.4
	c	0.9			5.3			1.2

 

With the statistical test, T Test, it was shown that Control and Filtered Control groups did not significantly differ. So these groups were pooled for further calculation.

 

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	1.459	1.295	7.4	1.796	1.822	4.8	1.972	2.097	3.5	1.742	1.738	15.0	23.8%
	b	1.361			1.679			2.152			1.730		23.0
	c	1.335			1.860			2.038			1.744		21.0
	d	1.281			1.926			2.004			1.737		22.8
	e	1.411			1.703			2.094			1.736		19.7
	f	1.361			1.842			1.965			1.723		18.5
Filtered Control	a	1.163			1.823			2.195			1.727		30.3
	b	1.131			1.786			2.275			1.731		33.2
	c	1.281			1.768			2.222			1.757		26.8
	d	1.281			1.815			2.172			1.756		25.5
	e	1.253			1.870			2.073			1.732		24.7
	f	1.224			1.995			2.006			1.742		25.7

CV: Coefficient of variation (%)

 

Table 6.1.5/3: Results of analysis – abiotic replicates

Nominal test item concentration	mg “OF14 AT”/L in algae solutions		Variation between T0 and T72 hrs (%)
	Results at T0 hour (deviation/nominal value)	Results at T 72 hours
Abiotic	< 5	< 5	NA
10 mg/L Abiotic	12.05 (+ 20.5 %)	11.35	- 5.8
102 mg/L Abiotic	95.3 (- 6.6 %)	150.6	+ 57.7
1050 mg/L Abiotic	937.4 (- 10.7 %)	1611.5	+ 71.9

NA: not applicable

 

Table 6.1.5/4: Results of analysis – biotic replicates

 

Nominal test item concentration	mg “OF14 AT”/L in algae solutions		Variation between T0 and T24 hrs (%)
	Results at T0  hour(deviation/nominal value)	Results at T 72 hours
Biotic	< 5	< 5	NA
10 mg/L Biotic	8.32 (- 16.8 %)	< 5 (3.76)	NA
102 mg/L Biotic	97.1 (- 4.8 %)	66.9	-31.1
1050 mg/L Biotic	1005.3 (- 4.3 %)	1364.1	+ 35.7

 

 

Nominal test item concentration	mg “OF14 AT”/L in algae solutions		Variation between T0 and T72 hrs (%)
	Results at T0 hour	Results at T 72 hours
1050 mg/L ABiotic(*)	937.4	1005.6	+ 7.3

(*) sample conserved at initial time (T0) in the fridge during 72 hours

 

The test item at nominal concentrations 102 and 1050 mg/L referenced Abiotic showed unexpected values because the test item stored at fridge during 72 h was considered as stable.

 

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 fulfilled.

Validity criteria fulfilled:

yes

Conclusions:

Under the test conditions, the 72 h ErC10, ErC50 of the test item “OF14 AT” were determined to be 33.1, 245.9 mg/L, respectively.

Executive summary:

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 OF14 AT 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 for the concentrations 328 and 1050 mg/L at 72 h in order to distinguish test substance particles from Pseudokirchneriella subcapitata alga cells.

 

The test design included 3 biotic replicates + 2 abiotic replicates at each test concentration, 6 replicates for the control and 6 biotic replicates + 2 abiotic replicates for the filtered control. Chemical analyses of test item were performed in biotic and abiotic replicates of algae test solutions.

 

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 fulfilled.

 

A concentration effect relationship was observed and statistically analyzed to obtain effect concentrations. The effect concentrations for the inhibition of growth rate ErC₅₀ and ErC₁₀ were 245.9 and 33.1 mg/L, respectively and the yield inhibition EyC₅₀ and EyC₁₀ were 67.1 and < 10 mg/L, respectively.

 

Under the test conditions, the 72 h ErC₁₀, ErC₅₀ of the test item “OF14 AT” was determined to be 33.1, 245.9 mg/L, respectively.

Description of key information

Under the test conditions, the 72 h ErC50 of the test item “OF14 AT” to the algae Pseudokirchneriella subcapitata was determined to be 245.9 mg/L.

Under the test conditions, the 72 h ErC10 of the test item “OF14 AT” to the algae Pseudokirchneriella subcapitata was determined to be 33.1 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

245.9 mg/L

EC10 or NOEC for freshwater algae:

33.1 mg/L