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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 June - 10 July 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results. However, since the maximal measured concentrations at the end of the test are in the range order of 0.01 mg/L and the lowest concentration for which the procedural recovery was determined was 0.1 mg/L, the study is considered reliable with restrictions (KC2). It has still been demonstrated that the concentrations to which the algae have been exposed was higher than the max. solubility of the substance and therefore the conclusion that the EC10 and EC50 are above the max. solubility in test medium remains valid.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
(2006; Annex 5 corrected 28 July 2011)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(2008, amended 2009)
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): IN-1000
- Appearance: Dark amber liquid
- Storage condition of test material: In refrigerator (2-8°C) container flushed with nitrogen

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Test concentrations were verified by chemical analysis. Water samples (2 mL, duplicate) were taken from the control and each exposure level at the start of the test and at t = 24h and t =72h.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Samples were stored in the freezer until analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
All test solutions were prepared separately and started with loading rates ranging from 1.0 to 100 mg/L. Two days of magnetic stirring was applied to reach the maximum solubility of the test substance in the test medium. The resulting aqueous mixtures were left to stabilize for 47 minutes where after the clear and colourless Water Accommodated Fractions (WAFs) were taken out by siphoning and used for testing.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture

ACCLIMATION
- Pre-culture: 3 to 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

After preparation, volumes of 50 mL test solution were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
24 mg CaCO3/L
Test temperature:
22 - 23 °C
pH:
7.9 - 8.1
Nominal and measured concentrations:
* Combined limit/range-finding test:
Blank control, WAFs were prepared at loading rates of 1.0, 10 and 100 mg/L
Analysed concentrations in WAFs at 1.0 and 100 mg/L: initial: 0.0096 and 0.90 mg/L, end: 0.0046 and 0.044 mg/L

*Final test:
Blank control, WAFs were prepared at loading rates of 1.0, 3.2, 10, 32, and 100 mg/L
The TWA measured concentrations of these WAFs were 0.059, 2.7, 15, 45 and 65 µg/L, respectively.

For calculation of TWA measured concentrations please refer to the formula presented under ‘attached background information’.
Details on test conditions:
TEST SYSTEM
- Test vessel:
100 mL all-glass capped vessels were used, each containing 50 mL of test preparation for the control and each treatment group.
All test vessels were maintained under identical conditions.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth which was diluted to a cell density of 1 x 10^4 cells per mL prior to use.

Replicates in final test:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels without algae per concentration (WAF at 10 mg/L) (replicates): 2
- 1 extra replicate of each test concentration and the control for sampling purposes

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., USA)

- Pre-culture and test medium: M2 according to OECD 201. The medium was prepared using reverse osmosis purified deionised water (Milli-RO, Millipore)

- Illumination: continuously using TLD-lamps with a light intensity within the range of 79-85 µE.m-2.s-1 while constantly shaking.

- Determination of cell concentrations: Samples were taken at 0, 24, 48 and 72 h. At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.

TEST CONCENTRATIONS
- Range finding study: A combined limit/range finding study was carried out
- Test concentrations: Algae were exposed to WAFs prepared at loading rates of 1.0, 10 and 100 mg/L and an untreated control
- Results used to determine the conditions for the definitive study: In the WAFs prepared at 1.0 and 10 mg/L no significant inhibition of the growth rate was observed. In the WAF prepared at a loading rate of 100 mg/L, 27% inhibition of the growth rate was observed.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (29 June - 2 July 2015)

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Basis for effect:
growth rate
Remarks on result:
other: The EC50 was > maximum soluble concentration of test substance in medium (TWA measured concentration 65 µg/L)
Duration:
72 h
Dose descriptor:
EC10
Basis for effect:
growth rate
Remarks on result:
other: The EC10 was > maximum soluble concentration of test substance in medium (TWA measured concentration 65 µg/L)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
45 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: growth rate: 1.3 mg/L (95% CI: 1.2-1.5 mg/L)
The 72h-ErC50 for the algal culture tested falls within the historical range at the test facility (0.82-2.3 mg/L).
Reported statistics and error estimates:
For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).
No EC50-values could be calculated because the test substance did not induce ≥50% effect on algal growth (EC50 > maximum soluble concentration tested).
Calculation of ECx values was based on probit analysis using linear maximum likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test substance.
The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Measured test substance concentrations (final test)

At the start of the test, the actual test concentrations were 6.0, 7.4, 31, 110 and 220 µg/L in the WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, respectively. These concentrations decreased to below the limit of detection (LOD = 0.519 µg/L) in the WAFs prepared at loading rates of 1.0 and 3.2 mg/L and to 14, 33 and 38 µg/L at the WAFs prepared at loading rates of 10, 32 and 100 mg/L, respectively, at the end of the test. Based on these results, the TWA measured concentrations were calculated (see Table 2). For the calculation of the effect parameters only the TWA concentrations obtained with the WAFs prepared at loading rates of 10, 32 and 100 mg/L were used.

Table 1: Concentrations of the test item in test medium

Time of sampling
[hours]

Loading rate1

[mg/L]

Concentration
analysed
[mg/L]

Relative to
initial
[%]

 

 

 

 

0

0

n.d.

 

 

1.0

0.006043

 

 

3.2

0.00744

 

 

10

0.0314

 

 

 102

0.0313

 

 

32

0.109

 

 

100

0.221

 

 

 

 

 

24

0

n.d.

 

 

1.0

n.d.

n.a.

 

3.2

0.004793

64

 

10

0.0126

40

 

 102

0.0123

39

 

32

0.0373

34

 

100

0.0507

23

 

 

 

 

72

0

n.d.

 

 

1.0

n.d.

n.a.

 

3.2

n.d.

n.a.

 

10

0.0135

43

 

 102

0.00811

26

 

32

0.03283

30

 

100

0.0381

17

 

 

 

 

1          A water accommodated fraction (WAF) prepared at the loading rate.

2          Without algae.

3          Estimated value, calculated by extrapolation of the calibration curve.

n.d.    Not detected. The limit of detection (LOD) was determined to be 0.519 µg/L.

n.a.     Not applicable.

Table 2:           TWA measured concentrations

IN-1000, WAF prepared at the given loading rate (mg/L)

Measured concentration (mg/L)

TWA (mg/L)

t=0h

t=24h

t=72 h

1.0

0.00604

<LOD

<LOD

0.00059

3.2

0.00744

0.00479

<LOD

0.0027

10

0.0314

0.0126

0.0135

0.015

101

0.0313

0.0123

0.00811

0.013

32

0.109

0.0373

0.0328

0.045

 100

 0.221

 0.0507

 0.0381

 0.065

1Without algae

Inhibition results

Growth rates were in the range of the control at TWA concentrations up to and including 45 µg/L during the 72-hour test period. Statistically significant inhibition of growth rate was found at a TWA exposure concentration of 65 µg/L.

Table 3: Percentage inhibition of growth rate during the final test

 

TWA conc.

IN-1000 (mg/L)

Mean

Std. Dev.

n

%Inhibition

Control

1.512

0.0491

6

0.00059

1.506

0.0561

3

0.4

0.0027

1.500

0.0425

3

0.8

0.015

1.513

0.0346

3

-0.1

0.045

1.468

0.0220

3

2.9

0.065

1.337

0.0846

3

11.6*

* - effect was statistically significant

Microscopic observations

Microscopic observations of the cells exposed to a WAF prepared at a loading rate of 100 mg/L at the end of the test revealed a normal and healthy appearance when compared to the control.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See 'Remarks on results including tables and figures'
Conclusions:
The ErC50 and ErC10 were > maximum soluble concentration of test substance in medium (TWA measured concentration 65 µg/L)
The NOErC was 45 µg/L (TWA measured concentration)
Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata, strain: NIVA CHL1. The method followed that described in OECD TG No 201. Pseudokirchneriella subcapitata was exposed to 5 WAFs prepared at loading rates between 1.0 and 100 mg/L (three replicate flasks per concentration) and a control (six replicate flasks) for 72 hours, under constant illumination and shaking at a temperature between 22 and 23 °C.

At the start of the test, the measured test concentrations were 6.0, 7.4, 31, 110 and 220 µg/L in the WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L, respectively. These concentrations decreased to below the limit of detection (LOD = 0.519 µg/L) in the WAFs prepared at loading rates of 1.0 and 3.2 mg/L and to 14, 33 and 38 µg/L at the WAFs prepared at loading rates of 10, 32 and 100 mg/L, respectively, at the end of the test. Based on these results, the TWA measured concentrations were calculated. For the calculation of the effect parameters only the TWA concentrations obtained with the WAFs prepared at loading rates of 10, 32 and 100 mg/L were used.

Growth rates were in the range of the control at TWA concentrations up to and including 45 µg/L during the 72-hour test period. Statistically significant inhibition of growth rate was found at a TWA exposure concentration of 65 µg/L. Inhibition of growth rate increased as exposure progressed at this test substance concentration.

The ErC50 and ErC10 were > maximum soluble concentration of test substance in medium (TWA measured concentration 65 µg/L)

The NOErC was 45 µg/L (TWA measured concentration).

Since the maximal measured concentrations at the end of the test are in the range of 0.01 mg/L and because the lowest concentration for which the procedural recovery was determined was 0.1 mg/L, the study is considered reliable with restrictions (KC2). It has still been demonstrated that the concentrations to which the algae have been exposed was higher than the max. solubility of the substance and therefore the conclusion that the EC10 and EC50 are above the max. solubility in test medium remains valid.