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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:

From 2018-03-12 to 2018-05-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Not relevant.

GLP compliance:

yes (incl. QA statement)

Remarks:

Signed on 2017-01-10

Specific details on test material used for the study:

None.

Analytical monitoring:

yes

Details on sampling:

Duplicate samples for analysis were taken from the control and the limit test loading rate (from a replicate of each test concentration without algae dedicated exclusively to chemical analyses) at the start and the end of the test. Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs. Concentration of Zirconium, a constituent of the test item, was also checked in the control medium and the limit test loading rate. TOC and Zirconium analyses were not performed in compliance with the OECD GLP principles but in accordance with ISO 17025.

Vehicle:

no

Details on test solutions:

The study was carried out using WAFs (Water Accommodated Fractions). The WAFs were prepared under closed conditions and by slow-stirring.
The mixing vessels were 1 L cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar was placed in each mixing vessel and test water was added. The loading rates of the test item were weighed on weighing boats that afterwards were placed above each mixing vessel and rinsed with test water. The mixing vessels were then carefully filled with the remaining volume of test water to obtain 1.1 L of test medium in order to use a maximum volume and to minimise headspace. Then the mixing vessels were closed immediately. The mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 23 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for at least 1 hour before use. The first 100 mL were discarded via the drain port. Then the WAFs were filtered and added into test vessels containing a fixed amount of inoculum (5.103 cells.mL-1 per vessel) that were immediately sealed after filling with a minimum headspace. At the start of the test, the solution was observed to be clear and colourless. The test was carried out without adjustment of the pH.
Controls: Test water without test substance but treated in the same way as the test substance solutions.

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, CCAP 278/4
Origin: Museum National d’Histoire Naturelle - 12, rue Buffon, Case N°19 - 75005 PARIS, bred in the Laboratoires des Pyrénées et des Landes under standardised conditions according to the test guidelines.
Reason for selection: This system is a unicellular algal species sensitive to toxic substances in the aquatic ecosystem and has been selected as an internationally accepted species.
Stock culture Algae stock cultures were started by inoculating growth medium (= test water) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 23 ± 2°C.

Test type:

static

Water media type:

freshwater

Remarks:

Original medium from OECD TG 201

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

None.

Post exposure observation period:

None.

Hardness:

No data.

Test temperature:

23±2 °C

pH:

Start(t=0 h): 8.00 to 8.04
End (t=72 h): 8.03 to 9.61

Dissolved oxygen:

No data.

Salinity:

No data.

Conductivity:

No data.

Nominal and measured concentrations:

Limit test: 100 mg/L (loading rate)

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass closed flasks with ground glass stopper, completely filled with test solution with minimum headspace.
Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Initial cells density: An initial cell density of 5.10^3 cells.mL-1 using the exponentially growing pre-culture.
- Replicates: 6 replicates for the control and the loading rate at 100 mg.L-1. Moreover, replicates of each treatment without algae were prepared for chemical analyses.

GROWTH MEDIUM
- Standard medium used: Yes; Original medium of OECD TG 201. Since the test was performed in sealed conditions, additional sodium bicarbonate was added to test water (for all treatments and inoculum suspension): 7 mL of NaHCO3 was added to the sterilised water during test water reconstitution (instead of 1 mL) to obtain a final concentration of 350 mg.L-1, according to the study plan.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: Continuous illumination
- Light intensity and quality: Continuous illumination with a light intensity of 4,440-8,880 lux and did not vary more than ± 15% from the average light intensity over the incubation area.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Cell numbers were counted daily by microscope using a counting chamber.
- pH: At the start and the end of the test in one vessel per concentration and the control (same vessel at t=0h and t=72h).
- Temperature of Medium: Measured continuously in the growth chamber, over the study period.
- Light Intensity: Light intensity was measured once (t=0 h) during the test at 5 positions distributed over the experimental area at the surface of the test media.

TEST CONCENTRATIONS
Limit test: 100.0 mg/L (loading rate)

RANGE FINDING STUDY
The range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1, 10, 32 and 100 mg.L-1 and to a control. No inhibition observed.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Details on results:

Based on the results of the range-finding test, a limit test was performed to demonstrate that the test item had no significant effect on Pseudokirchneriella subcapitata at the loading rate of 100 mg test item.L-1 under the experimental conditions or that EL50 was higher than 100 mg.L-1.
After 24 and 48 hours of exposure no significant inhibition of algal growth or yield relative to control values was observed. However, a slight inhibition was observed at t=72h, with 22.8% inhibition of growth rate. It should be noted that a significant number of microorganisms was observed during algae counting at the end of the test, but only at 100 mg.L-1 (not in controls), suggesting that this contamination was probably transmitted by the test item (the pre-culture and test vessels were sterile). This last observation could be the cause of the observed algal growth inhibition, following an impoverishment of test medium after 2 days.
Nevertheless, based on these results, the 48-h and 72h-ErL50, and the 48-h EyL50 were determined to be ≥ 100 mg.L-1.

Results with reference substance (positive control):

On February 26, 2018 (KA18-001; most recent test), the 72h-EC50 was 0.98 mg.L-1 for the parameter growth rate. Hence, the sensitivity of this batch of Pseudokirchneriella subcapitata was consistent with the level proposed by the ISO 8692 (6) (expected 72h-EC50: 0.92 mg.L-1 to 1.46 mg.L-1).

Reported statistics and error estimates:

The software ToxRat® Professional, version 3.2.1 was used to perform statistical analyses. Complementary statistical analyses were performed using another statistical spreadsheet (using Excel®) for the validity criteria of the study.

Biological results

Algal cell densities during the final test (expressed as density of algal cells.mL^-1x10⁴)

	Replicate	Nominal concentration (mg test item.L^-1)*
		Control	100
t=24h	1	4.0	3.6
	2	5.6	4.0
	3	6.0	4.8
	4	4.4	8.0
	5	6.4	4.0
	6	5.6	4.4
	Mean	5.3	4.8
	Std. Dev.	0.94	1.62
	% Inh.	-	5.5
t=48h	1	21.6	11.2
	2	18.4	15.6
	3	18.4	21.6
	4	19.6	16.4
	5	18.8	25.2
	6	28.0	16.0
	Mean	20.8	17.7
	Std. Dev.	3.73	4.95
	% Inh.	-	5.0
t=72h	1	90.4	18.8
	2	62.0	16.0
	3	80.0	44.0
	4	86.0	20.0
	5	72.4	33.6
	6	76.0	24.0
	Mean	77.8	26.1
	Std. Dev.	10.13	10.71
	% Inh.	-	22.8

* WAF prepared at the given loading rate.

The WAFs were filtered before addition into the test vessels.

At test start 5000algal cells.mL^-1were incubated.

Std. Dev.: standard deviation.

% Inh.: %Inhibition of growth rate relative to the control determined by the computer program ToxRat

Analytical results

Concentration of dissolved organic material and Zirconium (one of the main constituents of the test item) was checked in the controls and the loading rate of 100 mg.L^-1at the start and at the end of the test. Although every effort was made to maintain the concentrations of the WAFs (closed conditions with minimum headspace), TOC analyses indicated that organic compounds at 100mg.L^-1were maintained close to ± 30% of the initial concentration between the start and the end of the test. Moreover,analytical results indicated that very few Zirconium was found in the WAFs at 100 mg.L^-1. As observed in the daphnia study (LPL Project D18 -001), it was assumed that Zirconium from the test item sample was oxidised or eventually formed a precipitate since the first opening of the test item flask, and therefore was not easy to detect in this form with the analytical method. Despite these analytical results, the study was considered as valid given the results from the range-finding test which are consistent with the final test outcome, in agreement with the Study Monitor. In any case,since the test item was a multi-constituent substance, the results were based on nominal loading rates.

Concentrations of the test item in test water - TOC analysis results (mg.L^-1) - Final test 

	TOC (mg.L^-1)
Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	< 0.30	0.31
Control	< 0.30	0.30
100	32.7	22.0
100	32.3	22.5

* WAF prepared at the given loading rate.

LOQ_TOC: 0.30 mg Carbon.L^-1

Concentrations of Zirconium (mg.L^-1) in test water -Final test

 

Nominal concentration* (mg test item.L^-1)	Start (t=0h)	End (t=72h)
Control	< 0.01	< 0.01
Control	< 0.01	< 0.01
100	0.025	0.023
100	0.025	0.023

* WAF prepared at the given loading rate.

 

Physico-chemical parameter values throughout the test

Although the pH level in the control varied by more than 1.5 units at the end of the test (1.57 units of difference) this was not considered to have affected the integrity of the study. The cause of the pH increases in the controls and test concentrations was certainly due to the substantial algal growth in conjunction with closed conditions used in the test, despite the additionalsodium bicarbonate.

The mean light intensity was of 5049 lux (range: 4885-5220 lux), which remainedwithin the rangesprescribed by the study plan (range: 4440-8880 lux and shall not vary more than ± 15% from the average light intensity over the incubation area).

The temperature in the incubator was situated between 23.4 and 23.6°C throughout the test (average value: 23.5°C), and complied with the requirements as laid down in the study plan (23°C ± 2°C, constant within 2°C).

pH-values during the final test

	Nominal concentration(mg test item.L^-1)*
	Control	100
Start t=0h	8.04	8.00
End t=72h	9.61	8.03

* WAF prepared at the given loading rate.

Validity criteria of the study

Cell density in controls         156-fold increase within 72 hours. The specific growth rate was determined at 1.68 day^-1, so greater than 0.92 day^-1.

Coefficient of variation          1.The mean coefficient of variation for section-by-section specific growth rates in the control cultures was of 14.0%.

2.The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was of 2.7%.

Thus, all the validity criteria were fulfilled in this study.

 

Validity criteria fulfilled:

yes

Conclusions:

The toxic effect of the test item SOCOGEL PART I to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 for the parameters growth rate and yield were considered to be higher than 100 mg.L-1. The 72-hour EL50 for growth rate was also considered to be higher than 100 mg.L-1.

Executive summary:

A study was performed to assess the test item SOCOGEL PART I for its ability to generate toxic effects on the unicellular algal species Pseudokirchneriella subcapitata. The followed method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended by Commission Regulation (EU) 2016/266 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Based on the results of the range-finding test, a limit test was performed to demonstrate that the test item had no significant effect on the test organismunder the experimental conditionsor that EL₅₀was higher than 100 mg.L^-1. Algal cells were exposed to Water Accommodated Fractions (WAFs)of the test itemat a nominal loading rate of 100 mg test item.L^-1and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the control and the loading rate of 100 mg.L^-1was checked by TOC analysisat the startand the end of the test. Moreover, concentration of Zirconium (one of the main constituents of the test item) was also checked in the control and the limit test loading rate.

Although every effort was made to maintain the concentrations of the WAFs (closed conditions with minimum headspace), TOC analyses indicated that organic compounds at 100mg.L^-1were maintained close to ± 30% of the initial concentration between the start and the end of the test. Moreover,analytical results indicated that very few Zirconium was found in the WAFs at 100 mg.L^-1. After 24 and 48 hours of exposure no significant inhibition of algal growth or yield relative to control values was observed. However, a significant inhibition was observed at t=72h, with 22.8% inhibition of growth rate.

The toxic effect of the test item SOCOGEL PART I to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test usingWater Accommodated Fractions. Under the experimental conditions and based on nominal loading rates,the 48-hour EL₅₀for the parameters growth rate and yield was considered to be higher than 100 mg.L^-1. The 72-hour EL₅₀for growth rate was also considered to be higher than 100 mg.L^-1.

Results synopsis:

Test: Toxic effect to Pseudokirchneriella subcapitata according to OECD guidelines No. 201, using WAFs.

72h-EL50 grouth rate: > 100 mg/L

72h-EL50 yield: > 100 mg/L

 

Description of key information

The toxic effect of the test item SOCOGEL PART I to the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 for the parameters growth rate and yield were considered to be higher than 100 mg.L-1. The 72-hour EL50 for growth rate was also considered to be higher than 100 mg.L-1.

72h-EL50 grouth rate: > 100 mg/L

72h-EL50 yield: > 100 mg/L

Additional information

A study was performed to assess the test item SOCOGEL PART I for its ability to generate toxic effects on the unicellular algal speciesPseudokirchneriella subcapitata. The followed method was designed to be compliant with OECD Guideline for Testing of Chemicals No. 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test", referenced as Method C.3 of Commission Regulation No. 440/2008 amended byCommission Regulation (EU) 2016/266and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

Based on the results of the range-finding test, a limit test was performed to demonstrate that the test item had no significant effecton the test organismunder the experimental conditionsor that EL₅₀was higher than 100 mg.L^-1.Algal cells were exposed toWater Accommodated Fractions (WAFs)of the test itemat a nominal loading rate of 100 mg test item.L^-1and to a control. The inhibition of growth in relation to control cultures was determined over a test period of 72 hours, and thus over several algal generations.Concentration of dissolved organic material in the control and the loading rate of 100 mg.L^-1was checked by TOC analysisat the startand the end of the test. Moreover, concentration of Zirconium (one of the main constituents of the test item) was also checked in the control and the limit test loading rate.

Although every effort was made to maintain the concentrations of the WAFs (closed conditions with minimum headspace), TOCanalysesindicated that organic compounds at 100mg.L^-1weremaintained close to ± 30% of the initial concentration between the start and the end of the test. Moreover,analytical resultsindicatedthat very few Zirconium was found in the WAFs at 100 mg.L^-1.After 24 and 48 hours of exposure no significant inhibition of algal growth or yield relative to control values was observed. However, a significant inhibition was observed at t=72h, with 22.8% inhibition of growth rate.

The toxic effect of the test item SOCOGEL PART Ito the unicellular algal species Pseudokirchneriella subcapitata was investigated in a closed static test using Water Accommodated Fractions. Under the experimental conditions andbased on nominal loading rates,the 48-hour EL₅₀for the parameters growth rate and yield was considered to be higher than 100 mg.L^-1. The 72-hour EL₅₀for growth rate was also considered to be higher than 100 mg.L^-1.

Results synopsis:

Test: Toxic effect to Pseudokirchneriella subcapitata according to OECD guidelines No. 201, using WAFs.

72h-EL50 grouth rate: > 100 mg/L

72h-EL50 yield: > 100 mg/L