Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

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:
7th September 2018 to 13 October 23018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
Adopted March 23, 2006; Annex 5 corrected 28 July 2011
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Additiv 309
IUPAC Name:
Additiv 309
Test material form:
liquid: viscous
Details on test material:
Identification: Optimol Additiv 309
Appearance: Pasty dark brown liquid
Batch: 0000771660
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 27 June 2021 (expiry date)
Specific details on test material used for the study:
Test item information:

Identification: Optimol Additiv 309
Appearance: Pasty dark brown liquid
Batch: 0000771660
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 27 June 2021 (expiry date)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h, t=24 h and t=72 h.
Volume 2.0 mL from the approximate centre of the test vessels.
Storage Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the WAF prepared at a loading rate of 10 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Test solutions

Details on test solutions:
REPARATION OF TEST SAMPLES:
The substance tested was a pasty dark brown liquid UVCB which was not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item.

Preparation of test solutions started with loading rates individually prepared in the range of 1.0 and 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of two hours. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.

After preparation, volumes of 50 mL 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 10x4 cells/mL. Any residual volumes were discarded.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST SYSTEM:
Species: Raphidocelis subcapitata, strain: NIVA CHL 1

Source: In-house laboratory culture.

Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

FRESH WATER ALGAE CULTURE:
Stock culture: Algae stock cultures were started by inoculating growth medium 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 21-24°C.

Light intensity: 60 to 120 µE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.

Stock culture medium:
M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500mg/L
K2HPO4 39.5mg/L
MgSO4.7H2O 75mg/L
Na2CO3 20mg/L
C6H8O7.H2O 6mg/L
NH4NO3 330mg/L
CaCl2.2H2O 35mg/L
C6H5FeO7.xH2O 6mg/L
H3BO3 2.9mg/L
MnCl2.4H2O 1.81mg/L
ZnCl2 0.11mg/L
CuSO4.5H2O 0.08mg/L
(NH4)6Mo7O24.4H2O 0.018mg/L

Pre-culture: Three days before the start of the test, cells from the algal stock culture were inoculated in culture medium 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.

Pre-culture medium:
M2; according to the OECD 201 Guideline, formulated using Milli-RO water and with the following composition:
NH4Cl 15mg/L
MgCl2.6H2O 12mg/L
CaCl2.2H2O 18mg/L
MgSO4.7H2O 15mg/L
KH2PO4 1.6mg/L
FeCl3.6H2O 64µg/L
Na2EDTA.2H2O 100µg/L
H3BO3 185µg/L
MnCl2.4H2O 415µg/L
ZnCl2 3µg/L
CoCl2.6H2O 1.5µg/L
CuCl2.2H2O 0.01µg/L
Na2MoO4.2H2O 7µg/L
NaHCO3 50mg/L
Hardness (Ca+Mg) 0.24mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Study design

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

Test conditions

Hardness:
Hardness (Ca+Mg): 0.24mmol/L (24 mg CaCO3/L)
Test temperature:
During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21 24°C, constant within ±1°C).
pH:
8.1 ± 0.2
Details on test conditions:
TESTING STRATEGY AND EXPERIMENTAL DESIGN

COMBINED LIMIT/RANGE-FINDING TEST:
The project started with a combined limit/range-finding test. Six replicates of exponentially growing algae were exposed to a control and a WAF prepared at a loading rate of 100 mg/L, in the limit test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
• Three replicates per concentration were exposed to WAFs individually prepared at loading rates of 1.0 and 10 mg/L, in the combined range-finding test.
• Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
• pH was only measured in the control and the highest test concentration.
• At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

FINAL TEST:
Test concentrations:
Optimol Additiv 309: WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.
Control: Test medium without test item or other additives.
Replicates: 3 replicates of each test concentration,
6 replicates of the control,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae

TEST PROCEDURE AND CONDITIONS:
Test duration : 72 hours
Test type: Static
Test vessels: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
Test Medium: M2
Cell density: An initial cell density of 1 x 10-4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 70 to 78 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

MEASUREMENTS AND RECORDINGS:
pH: At the beginning and at the end of the test, for all test concentrations and the control
Temperature of medium: Continuously in a temperature control vessel.
Appearance of the cells: At the end of the final test microscopic observations were performed on the WAF prepared at a loading rate of 10 mg/L to observe for any abnormal appearance of the algae compared to the control.

RECORDING OF CELL DENSITIES:
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 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Test medium was used as blank and the extra replicates, without algae, as background for the treated solutions.




Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
8.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
4.1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
based on statistical significance
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
based on biological relevance
Key result
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks:
based on both statistical significance and biological relevance

Any other information on results incl. tables

RESULTS

Mean Cell Densities, Inhibition of Growth Rate and Inhibition of Yield

The mean cell densities measured during the combined limit/range-finding test are presented in Table 1.Table 2 and Table 3 present the percentages growth rate inhibition and yield inhibition per concentration, respectively.

Inhibition of algal growth increased with increasing loading rates of test item, reaching 100% growth rate inhibition and yield reduction at the WAF prepared at 100 mg/L.

Based on this result, samples taken from WAFs prepared at 1.0 and 100 mg/L were analysed. Separate analysis of components m/z 325.3and m/z 589.7was performed for each sample.

The actual test item concentrations based on the measurements of component m/z 325.3 were 0.020 and 2.4 mg/L in the samples taken from the WAFs prepared at 1.0 and 100 mg/L, respectively, at the start of the test. At the end of the test, the concentrations had decreased to 0.26 - 12% of initial according to the measured responses of component m/z 325.3.

The actual test item concentrations based on the measurements of component m/z 589.7were 1.8µg/L and 0.78 µg/L, respectively, at the start of the test. At the end of the test, the concentrations had decreased to 8.1-17% of initial according to the measured responses of component m/z 589.7.

All test conditions were maintained within the limits prescribed by the study plan.

Table 1          Mean Cell Densities (x104Cells/mL) during the Combined Limit/Range-Finding Test

Time (h) 

Optimol Additiv 309; Loading rate (mg/L)

Control

1.0

10

100

0

1.0

1.0

1.0

1.0

24

8.8

n.d.

n.d.

1.0

48

57.9

n.d.

n.d.

1.5

72

265

225

166

1.0

n.d. – not determined.


Table 2         Percentage Inhibition of Growth Rate during the Combined Limit/Range-Finding Test


Optimol Additiv 309
Loading rate (mg/L)
 

Mean

Std. Dev.

n

% Inhibition

Control

1.858

0.0412

6

 

1.0

1.805

0.0188

3

2.9

10

1.612

0.3477

3

13

100

0.000

0.0000

6

100

 

Table 3          Percentage Reduction of Yield during the Combined Limit/Range-Finding Test

Optimol Additiv 309
Loading rate (mg/L)
 

Mean

Std. Dev.

n

% Reduction

Control

264.1

32.17

6

 

1.0

223.7

12.62

3

15

10

165.1

111.80

3

37

100

0.0

0.00

6

100

 

Final Test                 

Samples taken from all test concentrations and the control were analysed. Separate analysis of components m/z 325.3and m/z 589.7was performed for each sample.

The actual test item concentrations based on the measurements of component m/z 325.3were 0.23, 0.62, 1.6, 2.1 and 12 mg/L at the WAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/L, respectively, at the start of the test. Measured test item concentrations increased with increasing loading rate of test item, confirming proper preparation of test solutions. During the 72-hour exposure period, the concentrations had decreased to 0.20 - 3.1% of initial according to the measured responses of component m/z 325.3 (see Table 4).

Measured concentrations based on component m/z589.7did not correlate to the loading rate of test item in both the combined limit/range-finding test as well as in the final test. Hence, it is unlikely that this component is responsible for the recorded toxicity towards algae. Actual test item concentrations based on component m/z 589.7can be found in Table 5

In view of the obtained results, it can be concluded that component m/z 325.3 might be responsible for the recorded effects on algal growth. Since the test item is a UVCB and test solutions were prepared as WAFs, the effect parameters were expressed in terms of loading rates.

Actual test item concentrations based on component m/z 325.3 in solutions incubated without algae were less stable than in their counterparts incubated with algae. However, since most concentrations were estimated by extrapolation of the calibration curve, no hard conclusions can be drawn on the influence of algae on the test item stability in test medium

Table 4          Loading Rates Versus Measured Concentrations based on Component m/z 325.3

Optimol Additiv 309

Loading rate (mg/L)

Measured concentration (mg/L)

t=0h

t=24h

t=72 h

Control

0.00322,3

0.00262,3

0.00262,3

1.0

0.23

0.022

0.00712

3.2

0.62

0.013

0.0093

10

1.6

0.019

0.00782

101

1.61

0.00481,2

0.00331,2

32

2.1

0.00562

0.00422

100

122

0.252

0.033

1without algae,2Estimated by extrapolation of the calibration curve,3recorded test item response was probably caused by carry-over.

Table 5          Loading Rates Versus Measured Concentrations based on Component m/z 589.7

Optimol Additiv 309

Loading rate (mg/L)

Measured concentration (mg/L)

t=0h

t=24h

t=72 h

Control

0.000372

0.0000432,3

0.000202,3

1.0

0.0088

0.0025

0.00088

3.2

0.029

0.00081

0.000372

10

0.025

0.000642

0.000132

101

0.0211

0.000251,2

0.000111,2

32

0.012

0.0000372

0.0000482

100

5.02

0.512

0.282

1without algae,2Estimated by extrapolation of the calibration curve,3recorded test item response was probably caused by carry-over.

Inhibition of Growth Rate and Inhibition of Yield   

Table 6 shows group mean growth rates and the percentages of growth rate inhibition (total test period) whereas Table 7shows the values at different time intervals. The group mean yields and the percentages of yield inhibition are summarized in Table 8.

Statistically significant growth rate inhibition and reduction of yield was found at loading rates of 3.2 mg/L and higher, reaching 100% inhibition at the WAFs prepared at 32 and 100 mg/L. However, growth rate inhibition recorded at the WAF prepared at 3.2 mg/L was below 10% and therefore considered to be biologically not relevant. Hence, the NOELR for growth rate inhibition was 3.2 mg/L based on biological relevance.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the WAF prepared at 10 mg/L when compared to the control.

Table 6        Growth Rate and Percentage Inhibition for the Total Test Period

Optimol Additiv 309
Loading rate (mg/L)
 

Mean

Std. Dev.

n

% Inhibition

Control

1.832

0.0215

6

 

1.0

1.834

0.0148

3

-0.11

3.2

1.711

0.0539

3

6.6#

10

0.699

0.2730

3

62*

32

0.000

0.0000

3

100*

100

0.000

0.0000

3

100*

* - effect was statistically significant,#effect was statistically significant however biologically not relevant (i.e. <10%).


Table 7          Growth Rate and Percentage Inhibition at Different Time Intervals

 

Optimol Additiv 309

Loading rate. (mg/L)

n

0 – 24 h

24 – 48 h

48 – 72h

Mean

% Inhibition

Mean

% Inhibition

Mean

% Inhibition

Control

6

2.139

 

1.879

 

1.479

 

1.0

3

2.231

-4.3

1.791

4.7

1.480

-0.11

3.2

3

1.917

10

1.705

9.2

1.512

-2.2

10

3

0.294

86

0.632

66

1.172

21

32

3

0.000

100

0.000

100

0.000

100

100

3

0.000

100

0.000

100

0.000

100


Table 8          Yield and Percentage Inhibition for the Total Test Period


Optimol Additiv 309
Loading rate (mg/L)
 

Mean

Std. Dev.

n

% Inhibition

Control

243.4

15.28

6

 

1.0

244.5

10.80

3

-0.47

3.2

170.3

28.72

3

30*

10

9.0

6.94

3

96*

32

0.0

0.00

3

100*

100

0.0

0.00

3

100*

* - effect was statistically significant.

Determination of Effect Concentration                 

Table 9 shows the effect parameters based on loading rates

Table 9          Effect Parameters

Parameter (mg/L)

NOELR*

NOELR#

EL10

EL20

EL50

Growth rate

Value

1.0

3.2

3.8

4.9

8.3

lower 95%-cl

 

 

2.8

3.9

7.5

upper 95%-cl

 

 

4.5

5.7

9.0

Yield

Value

1.0

1.0

2.2

2.7

4.1

lower 95%-cl

 

 

1.9

2.5

3.9

upper 95%-cl

 

 

2.4

2.9

4.5

cl – confidence limit, *based on statistical significance,#based on biological relevance.

Experimental Conditions                

Table 10 shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit).

During the exposure period the temperature measured in the incubator was maintained between 21 and 23°C. Temperature remained within the limits prescribed by the study plan (21‑24°C, constant within ±1°C).

Table 10        pH Levels Recorded during the Final Test

Optimol Additiv 309

Loading rate (mg/L)

pH

t=0h

t=72h

Control

8.0

8.4

1.0

8.0

8.3

3.2

8.0

8.3

10

7.9

8.1

32

7.9

8.1

100

7.9

8.1


 


 



Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of the present study with Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata), Optimol Additiv 309 inhibited growth rate of this fresh water algae species significantly at loading rates of 3.2 mg/L and higher.

The 72h-ECL50 for growth rate inhibition (ERL50) was 8.3 mg/L with a 95% confidence interval ranging from 7.5 to 9.0 mg/L.
The 72h-EL50 for yield inhibition (EYL50) was 4.1 mg/L with a 95% confidence interval ranging from 3.9 to 4.5 mg/L.
The 72h-NOELR for growth rate inhibition was 1.0 mg/L based on statistical significance.
The 72h-NOELR for growth rate inhibition was 3.2 mg/L based on biological relevance.
The 72h-NOELR for yield reduction was 1.0 mg/L based on both statistical significance and biological relevance.
Executive summary:

The objective ofthe study was to evaluate Optimol Additiv 309 for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) during an exposure period of 72 hours and, if possible, to determine the NOELR, EL10, EL20and EL50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in the report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2nd edition, Adopted July 6, 2018.

The batch of Optimol Additiv 309 tested was a pasty dark brown liquid UVCB which was not completely soluble in test medium at the loading rates initially prepared.Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging from 1.0 to 100 mg/L and used as test concentrations.

A final test was performed based on the results of a preceding combined limit/range-finding test.Six replicates of exponentially growing algal cultures were exposed to an untreated control, whereas three replicates per group were exposed to WAFs individually prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L. The initial algal cell density was 104cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Statistically significant growth rate inhibition and reduction of yield was found at loading rates of 3.2 mg/L and higher, reaching 100% growth rate inhibition and yield reduction at the WAFs prepared at 32 and 100 mg/L. However, growth rate inhibition recorded at the WAF prepared at 3.2 mg/L was below 10% and therefore considered to be biologically not relevant.

Samples taken from all test concentrations and the control were analysed.Separate analysis of components m/z 325.3and m/z 589.7was performed for each sample. The actual test item concentrations based on the measurements of component m/z 325.3 were 0.23, 0.62, 1.6, 2.1 and 12 mg/L at the WAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/L, respectively, at the start of the test. Measured test item concentrations increased with increasing loading rate of test item, confirming proper preparation of test solutions. During the 72-hour exposure period, the concentrations had decreased to 0.20 - 3.1% of initial according to the measured responses of component m/z 325.3.Similar as in the combined limit/range-finding test, measured concentrations based on component m/z589.7did not correlate to the loading rates initially prepared.

Since the test item is a UVCB and test solutions were prepared as WAFs, the effect parameters were expressed in terms of loading rates.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters based on loading rates are summarized in the table below.

Parameter (mg/L)

NOELR*

NOELR#

EL10

EL20

EL50

Growth rate

Value

1.0

3.2

3.8

4.9

8.3

lower 95%-cl

 

 

2.8

3.9

7.5

upper 95%-cl

 

 

4.5

5.7

9.0

Yield

Value

1.0

1.0

2.2

2.7

4.1

lower 95%-cl

 

 

1.9

2.5

3.9

upper 95%-cl

 

 

2.4

2.9

4.5

cl – confidence limit, *based on statistical significance,#based on biological relevance.