[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

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:

16/07/2018 - 19/07/2018

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0(control) and 100 mg/L
Samples were taken from the test solution and from the control solution at 24 hour intervals in order to better define loss of the test substance during the exposure period, based on the results of the analytical method validation. Analytical measurement was performed at the applied test concentration level and from the control at the beginning of the experiment and 24 hour intervals thereafter during the experiment. The samples were analyzed by HPLC - UV method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution with a nominal concentration of 100 mg/L was prepared with direct addition of the test item, mixed into the test medium (OECD Medium) using ultrasonic bath (7 minutes). As a Limit test was carried out, further dilution of stock solution was not performed.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Specie: Pseudokirchneriella subcapitata
- Strain: 61.81 SAG
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Germany.
- Method of cultivation: Algae are cultured under standardised conditions (OECD 201). The pre-culture was intended to give a quantity of algae suitable for the inoculation of test cultures. The pre-culture was prepared with the OECD algal growth medium, incubated under the same conditions as the test and used when still exponentially growing (after an incubation period of 4 days)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

22.7 – 22.8 ºC

pH:

7.21 – 8.95

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 100 mg/L.
Measured concentrations: 0 (control), 78.2 mg/L (geometric mean)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 250 mL Erlenmeyer flasks, continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.
- Aeration: No
- Initial cells density: 10E+04 algal cells per mL test medium
- Control end cells density: 69.5 x 10E+04 algal cells per mL test medium
- No. of vessels per concentration (replicates): six replicates
- No. of vessels per control (replicates): six replicates

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS: Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: Continuously illuminated.
- Light intensity and quality: 7594 lux (equivalent to ~103 μE/m2/s), which was ensured with fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber. Microscopic observation of the algal cells in test concentration level and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

TEST CONCENTRATIONS
Range finding study: A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control for 72h.
- Test concentrations: 0, 0.1, 1, 10 and 100 mg/L (2 replicates per each test concentration and 3 replicates in the control group, 72 hours)
- Results used to determine the conditions for the definitive study: Because no toxic effect was observed during the preliminary range-finding test, only one test concentration of the test item in the test medium (100 mg/L) and one control group were tested in a Limit Test.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate is tested at least twice a year by CitoxLab)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 78.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

78.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 78.2 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.:

> 78.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 78.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): There were no any observed morphological deviations during the experiment.

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
Potassium dichromate was tested at least twice a year to demonstrate satisfactory test conditions.
The date of the last study (Study Code: 18/178-022AL) with the reference item Potassium dichromate is (Batch Number: A0345704): 18 – 21 June 2018.
The 72h ErC 50: 0.99 mg/L, (95 % confidence limits: 0.91 – 1.07 mg/L)
The 72h EbC 50: 0.69 mg/L, (95 % confidence limits: 0.64 – 0.75 mg/L)
The 72h EyC 50: 0.60 mg/L, (95 % confidence limits: 0.55 – 0.65 mg/L)

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed to demonstrate exponential growth for the entire study period.
The inhibition of alga growth was determined from the biomass, the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel 2007 for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).
As a limit test was performed the ErC50, EbC50 and EyC50 values of the test item were not calculated and determined from the raw data.
Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (α = 0.05) by TOXSTAT software. For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (α = 0.05) by TOXSTAT software.

Table 2. Growth rates (µ) and Percentage Inhibition of µ during the Test Period.

Test group	Growth rate µ and % inhibition of µ
	0–24 h		0–48 h		0–72 h
	m	%	m	%	m	%
Control	0.0573*	0.0	0.0605	0.0	0.0591	0.0
78.2 mg/L (measured)	0.0553	3.5	0.0599	1.0	0.0590	0.2

*: at this value the rounding of the EXCEL and TOXSTAT software was different. The table contains the values calculated with EXCEL.

Table 3. Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period.

Test group	Areas under the Growth Curves (A) and % inhibition of A
	0–24 h		0–48 h		0–72 h
	A	%	A	%	A	%
Control	36.0	0.0	280.0	0.0	1320.0	0.0
78.2 mg/L (measured)	34.0	5.6	270.0	3.6	1298.0	1.7

Table 4. Yield (Y) and Percentage Inhibition of Y during the Test Period.

Test group	Yield (Y) and % inhibition of Y (0-72 h)
	Y	%
Control	69.3	0.0
78.2 mg/L (measured)	68.8	0.7

Table 5. pH-Values in the Test Media at the Start and End of the Test.

Test group	pH-values
	Start	End
Control	7.54	8.91
		8.87
		8.83
		8.95
		8.87
		8.90
78.2 mg/L (measured)	7.21	8.41
		8.51
		8.53
		8.47
		8.44
		8.50

Table 6. Temperature in the Climate Chamber and in the Test Media during Test.

Parameter		Exposure Time
		Day 0	Day 1	Day 2	Day 3
temperature (ºC) measured in flask		22.8	22.7	22.8	22.7
temperature (ºC) of the climate chamber	max	22.9	23.0	23.2	23.0
	min	22.2	22.4	22.6	22.3

Table 7. Cell number (xE4 cell/mL) determined in the Main Experiment.

Test group		Number of cells
		0 h	24 h	48 h	72 h
Control		1	4	16	70
		1	4	19	74
		1	5	17	67
		1	3	20	71
		1	4	18	68
		1	4	20	72
	Mean	1.00	4.00	18.33	70.33
	SD	0.0	0.6	1.6	2.6
78.2 mg/L (measured)		1	5	16	69
		1	4	20	74
		1	3	18	68
		1	3	16	67
		1	4	20	72
		1	4	17	69
	Mean	1.00	3.83	17.83	69.83
	SD	0.0	0.8	1.8	2.6

Validity criteria fulfilled:

yes

Remarks:

(cell density in control increased by the factor of 70.33>16, mean coefficient of variation for section-by-section specific growth rates in control was 12.13%<35%, coefficient of variation of average specific growth rates in control was 0.86%<7%)

Conclusions:

The 72h-EC50 in green algae Pseudokirchneriella subcapitata was found to be higher than 78.2 mg/L and the NOEC was determined to be 78.2 mg/L (measured values), both based on growth rate.

Executive summary:

The Growth inhibition test on Algae (Pseudokirchneriella subpicata) was performed in accordance with OECD Guideline 201 and EU Method C.3. The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a semi-static system. Based on the performed preliminary range-finding test, a Limit Test was carried out using only one test concentration of 100 mg/L (nominal) and one control group. The test design included six replicates at test concentration and six replicates for the untreated control. All validity criteria were met. The test concentrations were analytically determined at 24 hour periods. The biological results are based on the measured geometric mean test item concentration; the decrease of the test item may have been caused by a biological effect related to the test solutions in presence of living test system. The EC50 were determined to be > 78.2 mg/L (geometric mean, basis for effect: growth rate, yield and biomass). The overall NOEC was determined to be 78.2 mg/L (geometric mean) and the overall LOEC was determined to be > 78.2 mg/L (geometric mean).

Description of key information

Key study. Method according to OECD 201 (limit test), GLP study. The EC50 were determined to be > 78.2 mg/L (nominal, basis for effect: growth rate, yield and biomass). The overall NOEC was determined to be 78.2 mg/L (nominal) and the overall LOEC was determined to be > 78.2 mg/L (nominal).

Key value for chemical safety assessment

EC50 for freshwater algae:

78.2 mg/L

EC10 or NOEC for freshwater algae:

78.2 mg/L

Additional information

Key study. The Growth inhibition test on Algae (Pseudokirchneriella subpicata) was performed in accordance with OECD Guideline 201 and EU Method C.3. The effect of test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata, over an exposure period of 72 hours in a semi-static system. Based on the performed preliminary range-finding test, a Limit Test was carried out using only one test concentration of 100 mg/L (nominal) and one control group. The test design included six replicates at test concentration and six replicates for the untreated control. All validity criteria were met. The test concentrations were analytically determined at 24 hour periods. The biological results are based on the measured geometric mean test item concentration; the decrease of the test item may have been caused by a biological effect related to the test solutions in presence of living test system. The EC50 were determined to be > 78.2 mg/L (geometric mean, basis for effect: growth rate, yield and biomass). The overall NOEC was determined to be 78.2 mg/L (geometric mean) and the overall LOEC was determined to be > 78.2 mg/L (geometric mean).