β-methyl-3-(1-methylethyl)benzenepropanal

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

13 December, 1990 - 21 June, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

A more recent OECD 202 on FLORHYDRAL has been performed since this 1991 study.

The study described here has a number of short-comings and should only be used as representative supporting evidence :
- Limited number of analyses performed at only selected test concentrations and selected timepoints;
- Large variance in % of nominal vs actual measured concentrations at fresh and also aged exposure solutions;
- Florhydral is a Primary Aldehyde and subject to rapid auto-oxidation to the alcohol- and carboxylic-acid forms when in aerated water, thus, necessitating at least semi-static renewal conditions and, moreover, closer analytical monitoring at all fresh and aged time-points for Parent, and, eventually, the corresponding Alcohol- and Carboxylic Acid-Metabolites.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch No. 183831;
Purity 97.3%;
Date of Expiry : 30 April, 1991

Analytical monitoring:

yes

Details on sampling:

Analysis of Water Samples from Main test :
Fresh samples at Time 0 and Aged samples at 24 hours were sampled and analysed in duplicate for the Solvent Control, 0.625 mg/L, 2.5 mg/L and 10.0 mg/L Nominal test concentrations.

Samples of the 1.25 mg/L and 5.0 mg/L Nominal test concentrations were not analysed.

Vehicle:

yes

Remarks:

0.01% Acetone as carrier solvent.

Test organisms (species):

Daphnia magna

Details on test organisms:

D. magna bred in the testing facility. Breeding was performed in 10L glass vessels in mixtures of pond water and reconstituted water with slight aeration. They were fed with algae and kept under the same temperature and light conditions as in the main test tself. For the test, animals with an age less than 24 hours old were used.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water.

Limit test:

no

Total exposure duration:

48 h

Test temperature:

19.5°C

pH:

pH 7.6 at the start of exposure
pH 8.0 at the end of the first renewal period.

Dissolved oxygen:

9.6 mg O2/L at beginning of the exposure
9.2 mg O2/L afater 24 hours exposure.

Nominal and measured concentrations:

Analysis of Time 0 samples indicated exposure concentrations of between 69.9% and 118.9% of Nominal.

Analysis of 24 hour aged exposure solutions illustrated between 30.5% and 72.2% of Nominal concentration remaining.

Details on test conditions:

Exposure : 50 mL beakers, containing 20 mL of exposure solution and 10 daphnids each, in duplicate;
Lighting : 16 hour / 8 hour light/dark cycle (between 500 - 2000 Lux);
Static Renewal : Renewal of exposure solutions after 24 hours;

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

5.48 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Measured concentration based on 71.2% recovery Time 0 to 24 hours.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

7.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

Potassium Dichromate :
24 hour EC50 = 1.608 mg/L
48 hour EC50 = 1.247 mg/L

24 hour EC0 = 0.804 mg/L
48 hour EC0 = 0.623 mg/L

Validity criteria fulfilled:

yes

Conclusions:

For D. magna exposed to FLORHYDRAL :
48 hour EC50 = 7.70 mg/L based on Nominal test concentrations
48 hour EC50 = 5.48 mg/L based on 71.2% recovery Time 0 to 24 hours.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May, 2016 - 14 June, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

FLORHYDRAL :
Batch No. : SC00016096
Purity : 99.3%
Expiry Date : 29 August, 2016

Analytical monitoring:

yes

Details on sampling:

Test item concentration in test solution :
It was confirmed that the converted product (GR-81-6079 – Florhydral Acid) of test item was generated in the test solution at 24 hours after during exposure in preliminary study. Therefore, the concentration of test item and converted products (GR-81-6079 and GR-82-4337 – Florhydral Alcohol) were measured in the definitive study.
Frequency of measurement At the start of exposure, before and after the renewal of test solution, and the end of exposure
Sample for measurement Another solution sampled separately from the preparation container (at the preparation)
One test vessel in each test level (before the renewal of test solution and at the end of exposure)
Volume of sample Approximately 50 mL (all test levels).

Vehicle:

no

Details on test solutions:

Preparation of test solution :
The test sample (0.0110 g) was added into an Erlenmeyer flask filled with dilution water (1.1 L) to produce 10.0 mg/L as nominal concentration. The flask was immediately sealed with a plug, and then stirred for 1 hour with a magnetic stirrer to prepare the stock solution. Required amount of the stock solution and dilution water were mixed and stirred in a preparing container to prepare the test solution which was divided into each test vessel. In addition, the preparation was repeated twice a test.

Test concentration 8.00, 4.71, 2.77, 1.63 and 0.958 mg/L (a geometric series with a factor of 1.7)
The test concentrations and the geometric factor were decided from the results of a preliminary study.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test organisms :
Species Daphnia magna Clone A
Reason for selection of species Species recommended in the test guidelines
Source of supply The University of Sheffield, UK
Date of supply July 9, 1990
Acclimation Young neonate daphnids (< 24 hours old) produced by parents that were cultured in this laboratory were used. The parents to obtain young daphnids were bred in the same quality of water (dechlorinated tap water), water temperature (20±1°C) and photoperiod (16-hour light/8-hour dark) as used in the test. The parent animals used for the test were same lot and their age and survival rate were 20-day old and 100%, respectively. Chlorella vulgaris of 0.1-0.2 mgC (Organic carbon content)/day per Daphnia was fed to the parents once a day.
Selection of young daphnids Less than 24-hour-old daphnids
Allocation to the test groups Random samplinng.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Dechlorinated tap water, aerated sufficiently and temperature-controlled, was used.

Limit test:

no

Total exposure duration:

4 h

Hardness:

37 mg/L as CaCO3

Test temperature:

19.7 to 20.3 °C

pH:

pH 7.7 to 7.8

Dissolved oxygen:

8.3 to 8.4 mg O2/L

Conductivity:

15 mS/m

Nominal and measured concentrations:

The Nominal concentrtions tested were :
Control
0.958 mg/L
1.63 mg/L
2.77 mg/L
4.71 mg/L
8.00 mg/L

The Freshly prepared exposure solutions (Time 0 and 24 hour renewal) were between 81.9 to 99.3% of Nominal. The aged, 24 hour old exposure solutions contained between 80.3 and 95.8% of the Nominal Parent test material, thus demonstrating good stability over the 24-hour period.

The Nominal vs Geometric Mean Measured exposure concentrations were established to be, as follows :

Nominal (mg/L) Geometric Mean (mg/L)
Control Not Detected (<0.07 mg/L)
0.958 0.830
1.63 1.44
2.77 2.47
4.71 4.17
8.00 7.27

In addition to analysis for Parent material,quantification of the Florhydral-Acid (GR-81-6079) and Florhydral-Alcohol (GR-82-4337) was also performed.
GR-81-6079 - In Fresh exposure solutions, only detected in the highest test concentration level at an amount equivalent to 1.01% of the theoretical dose. Only small quantities were determined in the 24-hour old water samples at the two highest exposure solution levels, constituting between 1.58 and 2.02% of the theoretical dose.

Details on test conditions:

Test conditions :
Type of test Semi-static regime (renewal at 24 hours after), closed system without headspace
Exposure duration 48 hours
Test concentration 8.00, 4.71, 2.77, 1.63 and 0.958 mg/L (a geometric series with a factor of 1.7)
The test concentrations and the geometric factor were decided from the results of a preliminary study.
Control Dilution water without the test item
Replicate 4 replicates/test level
Number of organism 20 daphnids/test level (5 daphnids/test vessel)
Volume of test solution Approximately 200 mL/test level (approximately 50 mL/test vessel)
Temperature of test solution 20±1°C
Dissolved oxygen concentration More than 3 mg/L without aeration
pH adjustment No adjustment
Lighting condition Room light, 16-hour light/8-hour dark
Feeding No feeding.

Reference substance (positive control):

yes

Remarks:

Potassium DiChromate

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: All analyses of exposure solutions demonstrated > 80% of Nominal exposure concentration, therefore, 48 hour EC50 reported as Nominal. 95% CI : 1.99 - 2.53 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI : 1.77 - 2.25 mg/L

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0.958 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0.83 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

4.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

4.17 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Immobility :
The minimum concentration causing 100% immobility at 48 hours was 4.71 mg/L. Maximum concentration causing no immobility at 48 hours was 0.958 mg/L. Immobility in the control during exposure was 0%, which meets the criterion for the validity of the test (i.e. not more than 10%).

Observed abnormal response :
Observed abnormal responses during exposure were lethargy and immobilization.
In the control, no abnormal response (discolour of body, trapping at the surface of the water and so on), which met the criterion for the validity of the test (i.e. not more than 10%), was observed during exposure. No effects were observed on the daphnids at the 0.958 mg/L (nominal) test concentration.

Results with reference substance (positive control):

Confirmation of reproducibility of test system A 48-hour acute immobilization test of a reference substance with the test organisms was periodically conducted. The latest data is shown below.
Reference substance Potassium dichromate (JIS special grade, Wako Pure Chemical Industries, Ltd. Lot No. RCK7838)
Period of study April 12 - April 14, 2016
48-hour EC50 0.14 mg/L
This value was within the stipulated range (mean ± 2S.D.) [mean ± S.D.: 0.23±0.06 mg/L (n=96)] to background data in this laboratory.

Reported statistics and error estimates:

Calculating method of EC50 :
The 48-hour EC50 value was calculated by Probit analysis, and also the 95% confidence limits and the slope of the concentration-response curve were calculated. For 24 hours, the EC50 was calculated by Binomial test.

The EC50 was estimated by using Computer Program (running on Microsoft software "Excel") developed by our laboratory.

The results of this study were estimated based on nominal concentrations since the measured concentrations of test item in test solution were maintained within ±20% of the nominal concentrations during exposure. In addition, the EC50s based on the geometric mean measured concentration of test item and the based on the total measured concentration of test item and converted products (calculated as amount of test item) was also estimated as a reference value.

Immobility :

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the following end-points were derived for D. magna exposed to FLORHYDRAL according to OECD TG 202 :
48 hour EC50 = 2.26 mg/L
48 hour EC0 = 0.958 mg/L
48 hour EC100 = 4.71 mg/L

This study was conducted in order to confirm the effect of the test item on the test organisms below the solubility of the test item in dilution water. As a result, 48-hour EC50 was 2.26 mg/L. Since the test item has slight volatility and oxidizability, closed vessel was used in this study. As the result, the measured concentrations of the test item in the test solution were within the range of ±20% of the nominal concentration during the exposure.

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 March, 2015 - 21 July, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

FLORHYDRAL is a primary aldehyde and undergoes transformation to the following two Primary Degradates :
- Florhydral-Acid (GR-81-6079), and
- Florhydral-Alcohol (GR-82-4337)
Both of the above primary metabolites have been observed in an OECD 301C study, and were also observed to have been formed in an OECD 203 study.


In the OECD 301C, on Day 28 72-77% of the starting dose had been transformed in to Florhydral-Acid, and 18 - 20% was present as the Florhydral-Alcohol. Both entities, and in particular the Florhydral-Acid are relevant for consideration within environmental studies.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-81-6079 (Florhydral-Acid) :
Lot No. : GLN00538-026-DF
Purity : 97.0%
Date of Expiry : 22 July, 2015

Analytical monitoring:

yes

Details on sampling:

During a pre-study phase, it was observed that the GR-81-6079 was relatively stable under the test conditins and for the duration of 48 hours. As such, a static set-up was performed.

Samples of the Control and all five exposure concentration levels were analysed at Time 0 (Fresh) and at the end of the exposure phase at 48 hours (aged samples).

Vehicle:

no

Details on test solutions:

Based on the results of a preliminary non-GLP range finding test, the following nominal
concentrations in a geometrical series (spacing factor: 2.2) were tested in the definitive test:
100, 45.5, 20.7, 9.39, and 4.27 mg test item/L.

Additionally, daphnids were exposed under control conditions (non-treated test medium).

The test item was applied once at the beginning of the test. Four replicates were used for the
test item concentrations, and for the control(s). Each replicate vessel contained five
daphnids.

The test was performed over 48 hours under static conditions.

Preparation of Stock and Test Solutions :
A stock solution (SL) of the test item was prepared by dissolving 100 mg of the test item in
1000 mL of Medium M4 (nominal concentration 100 mg test item/L, identical with the highest
test concentration, C5) using vigorous stirring with 2 times intermittent ultrasonication. This
stock solution was stirred for 4.5 hours before first use. It was used thereafter to prepare the test
item concentration levels by diluting corresponding volumes of the test item stock solution with
Medium M4. These test solutions were used to pre-condition the test vessels overnight. A
second batch of the stock solution was prepared on the same day, stirred overnight at ambient
temperature in the dark and used to prepare a second set of test item concentration levels for
exposure of the test system.

The stock solution was free of undissolved/particulate matter as determined by visual
inspection.

Corresponding volumes of SL (=C5) were taken and diluted with Medium M4 to prepare the
test solutions.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System :
 Species: The test system used in this study was Daphnia magna STRAUS (clone M10), a
water flea, cultured at ECT Oekotoxikologie GmbH since December 22, 2011.
 Origin: The organisms were originally supplied by KU Leuven, Belgium

Holding Conditions :
The culture conditions of Daphnia magna as summarised below are similar to the test
conditions.
Material of stock vessel: glass
Volume of medium per stock vessel: 1.8 L
Depth of medium in the stock vessel: approximately 14.5 cm
Number of daphnids kept as stock per culture vessel (batch size): approximately 20
Separation of adult and young daphnids: 1 to 5 times per week
Medium: Elendt medium M4
Renewal of medium: twice per week
Temperature: 20  2°C
Photoperiod (light/dark) 16/8 h
Light intensity: 50 to 1000 lx
Aeration: none
Food: Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
Feeding frequency: 4 times per week.

Test type:

static

Water media type:

freshwater

Remarks:

Elendt medium M4 was used to dilute the test item and to keep the daphnids during the period of the test.

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

20.0 - 21.0°C

pH:

pH 7.2 - 7.8

Dissolved oxygen:

8.4 - 8.8 mg O2/L

Conductivity:

674 microS/cm

Nominal and measured concentrations:

Freshly prepared Time 0 exposure solutions were between 66.8 to 92.3 % of the Nominal exposure concentrations. Following 48 hour, the exposure concentrations were between 73.6 and 86.0% of Nominal, demonstrating good stability.

The Nominal and Geometric Mean measured concentrationsdetermined are, as follows :

Nominal Geometric Mean Measured
Control Not Detected (< 0.45 mg/L)
4.27 mg/L 3.17 mg/L
9.39 mg/L 7.62 mg/L
20.7 mg/L 18.4 mg/L
45.5 mg/L 34.0 mg/L
100 mg/L 81.2 mg/L

Details on test conditions:

Exposure Conditions :
Test organism: Daphnia magna (Straus), clone M10
Age of parental daphnids: less than 35 d
Age of the daphnids at the beginning of the test: less than 24 h
Test units: screw-cap glass vessels (nominal volume: 60 mL)
covered by watchglasses; pre-conditioned overnight
Volume of test solution per test vessel: 50 mL
Test medium: Medium M4 (OECD 2004)
Adjustment of pH in the medium: none
Application of test item: test item dissolved in Medium M4 applied once at the beginning of the test
Test duration (exposure): 48 h
Aeration of test vessels: none
Renewal of the test solution during the test period: none
Feeding during exposure: none
Temperature (nominal): 18 to 22°C, constant within a range of 2°C
Light regime: 16 h light : 8 h dark
Light intensity: 335 to 348 lx (n = 3)
Biological parameter: immobility
Sampling for chemical analysis of test item concentrations:at start and at end of exposure

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

47.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI : 39.8 - 58.9 mg/L

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

18.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 81.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Biological Results :
After 48 hours exposure, 90% and 20% of the daphnids were observed to be immobile at the highest (Nominal 100 mg/L) and second highest (Nominal 45.5 mg/L) exposure concentration. No daphnids were observed to be immobile during the 48 hour study at the Control and 4.27, 9.39 and 20.7 mg/L Nominal test concentrations.

Sublethal observations on the daphnids were concentration-related. The frequency of
daphnids that were trapped on the water surface, less active than the controls or lying on the
bottom of the test vessel was increased at 20.7, 45.4 and 100 mg/L. At 4.27 and 9.39 mg/L
the daphnids behaved like the control animals.

Results with reference substance (positive control):

The reference test with potassium dichromate performed in September 2014 resulted in an
EC50(24 h) of 1.11 mg/l.

According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for
potassium dichromate obtained from different laboratories should range between 0.6 mg/l
and 2.1 mg/l. The toxicity of the reference item is within this range. Therefore the results of
this reference test are acceptable and the test conditions are reliable and the sensitivity of
the test system could be demonstrated.

Reported statistics and error estimates:

Data Assessment and Statistical Evaluation :
Mortality as assessed by immobility of the daphnids was evaluated. Probit analysis using
linear maximum likelihood regression was used to estimate the ECx values. Inhibitions lower
or equal to 0% or greater or equal to 100% were replaced by 0.10 and 99.9, respectively.
Fisher's Exact test (p≤0.05) was used to determine the threshold concentrations for daphnid
immobility.

The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH,
Naheweg 15, D-52477 Alsdorf) was used for these calculations.

The biological endpoints were expressed based on the nominal and on mean measured
concentrations.

Immobility :

Validity criteria fulfilled:

yes

Conclusions:

The results of an OECD 202 study performed with Florhydral-Acid (GR-81-6079) with Daphnia magna gave the following :
48 hour EC50 = 47.6 mg/L
48 hour EC0 = 20.7 mg/L
48 hour EC100 > 81.2 mg/L

Reference 4

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

25 March, 2015 - 21 July, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

FLORHYDRAL is a primary aldehyde and undergoes transformation to the following two Primary Degradates :
- Florhydral-Acid (GR-81-6079), and
- Florhydral-Alcohol (GR-82-4337)
Both of the above primary metabolites have been observed in an OECD 301C study, and were also observed to have been formed in an OECD 203 study.

In the OECD 301C, on Day 28 72-77% of the starting dose had been transformed in to Florhydral-Acid, and 18 - 20% was present as the Florhydral-Alcohol. Both entities, and in particular the Florhydral-Acid are relevant for consideration within environmental studies.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

April, 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-82-4337 (Florhydral-Alcohol) :
Lot No. : GLN00538-034-Fr10+11
Purity : 98.7%
Date of Expiry : 22 July, 2015

Analytical monitoring:

yes

Details on sampling:

A preliminary range-finding study demonstrated that under the test conditions the test item was stable for the 48 hour duration. As such, a static exposure system was adopted for the final, main test.

Samples of the Control and the five exposure concentration levels were aken at the Start of the Exposure (0 hours) and at the end of the exposure (48 hours) for substance-specific analytics.

Vehicle:

no

Details on test solutions:

Based on the results of a preliminary non-GLP range finding test, the following nominal
concentrations in a geometrical series (spacing factor: 2.2) were tested in the definitive test:

50.0, 22.7, 10.3, 4.70, and 2.13 mg test item/L

Additionally, daphnids were exposed under control conditions (non-treated test medium).

A stock solution (SL) of the test item was prepared by dissolving 100 mg of the test item in 2000
mL of Medium M4 (nominal concentration 50 mg test item/L, identical with the highest test
concentration, C5). This stock solution was stirred for 1 hour, ultrasonicated for 5 min, stirred for
1.5 h, and ultrasonicated for 5 min before first use. This treatment was performed in the dark. It
was used thereafter to prepare the test item concentration levels by diluting corresponding
volumes of the test item stock solution with Medium M4. These test solutions were used to pre-condition the test vessels overnight. A second set of test item concentration levels was prepared
from the stock solution (after stirring overnight at ambient temperature in the dark) for exposure
of the test system.

The stock solution was free of undissolved/particulate matter as determined by visual
inspection.

Corresponding volumes of SL (=C5) were taken and diluted with Medium M4 to prepare the
test solutions. The following table describes the procedure for preparing the test solutions, and
the control for the test.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test System :
 Species: The test system used in this study was Daphnia magna STRAUS (clone M10), a
water flea, cultured at ECT Oekotoxikologie GmbH since December 22, 2011.
 Origin: The organisms were originally supplied by KU Leuven, Belgium.

Holding Conditions :
The culture conditions of Daphnia magna as summarised below are similar to the test
conditions.
Material of stock vessel: glass
Volume of medium per stock vessel: 1.8 L
Depth of medium in the stock vessel: approximately 14.5 cm
Number of daphnids kept as stock per culture vessel (batch size): approximately 20
Separation of adult and young daphnids: 1 to 5 times per week
Medium: Elendt medium M4
Renewal of medium: twice per week
Temperature: 20 +/- 2°C
Photoperiod (light/dark) 16/8 h
Light intensity: 50 to 1000 lx
Aeration: none
Food: Algae (Desmodesmus subspicatus), instant baker’s yeast suspension, TetraMin® suspension
Feeding frequency: 4 times per week.

Test type:

static

Water media type:

freshwater

Remarks:

Elendt M4

Limit test:

no

Total exposure duration:

48 h

Hardness:

250 mg/L as CaCO3

Test temperature:

19.7 - 20.1 °C

pH:

pH 7.3 - 7.4

Dissolved oxygen:

8.7 - 9.0 mg O2/L

Conductivity:

674 microS/cm

Nominal and measured concentrations:

Analysis of Time 0 exposure solutions demonstrated a recovery of between 95.6 - 105% with respect to Nominal.

Analysis of the samples following 48 hours of exposure demonstrated recoveries of between 87.8 and 96.5% of Nominal, thus, illustrating stability in the test system.

No detects at or above1/3 of the LOQ were observed in the Control samples analysed.

Nominal Concn. (mg/L) Geometric Mean Measured Concn. (mg/L)
Control Not Detected (< 0.3 mg/L)
2.13 2.04
4.70 4.33
10.3 9.99
22.7 22.2
50.0 46.6

Details on test conditions:

Exposure Conditions :

Test organism: Daphnia magna (Straus), clone M10
Age of parental daphnids: less than 35 d
Age of the daphnids at the beginning of the test: less than 24 h
Test units: screw-cap glass vessels (nominal volume: 60 mL)
covered by watchglasses; pre-conditioned overnight
Volume of test solution per test vessel: 60 mL
Test medium: Medium M4 (OECD 2004)
Adjustment of pH in the medium: none
Application of test item: test item dissolved in Medium M4 applied once at the
beginning of the test
Test duration (exposure): 48 h
Aeration of test vessels: none
Renewal of the test solution during the test period: none
Feeding during exposure: none
Temperature (nominal): 18 to 22°C, constant within a range of 2°C
Light regime: 16 h light : 8 h dark
Light intensity: 279 to 386 lx (n = 3)
Biological parameter: immobility
Sampling for chemical analysis of test item concentrations: at start and at end of exposure

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

12.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI : 9.85 - 15.0 mg/L. All exposure solutions were maintained at > 80% of Nominal. As such, main result is reported with respect to Nominal test concentrations.

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

11.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI : 9.45 - 14.6 mg/L

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

4.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

4.33 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

50 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

46.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Sublethal observations on the daphnids were few, and not clearly concentration-related.
Daphnids that were trapped on the water surface or less active than the controls were
observed (0-2 individuals per replicate) across all concentration levels (C1-C5), but a clearly
concentration-related pattern was not recognised. Otherwise the daphnids generally behaved
like the control animals.

Results with reference substance (positive control):

The health of the Daphnia culture and the overall quality of the test organisms is monitored
regularly by using the reference toxicant potassium dichromate (K2Cr2O7) in an acute test
design in order to determine the sensitivity of the organisms from the culture and to
demonstrate that the test conditions are appropriate.

The reference test with potassium dichromate performed in September 2014 resulted in an
EC50(24 h) of 1.11 mg/l.

According to the OECD guideline No. 202 (OECD, April 2004) EC50(24 h)-values for
potassium dichromate obtained from different laboratories should range between 0.6 mg/l
and 2.1 mg/l. The toxicity of the reference item is within this range. Therefore the results of
this reference test are acceptable and the test conditions are reliable and the sensitivity of
the test system could be demonstrated.

Reported statistics and error estimates:

Data Assessment and Statistical Evaluation :
Mortality as assessed by immobility of the daphnids was evaluated. Probit analysis using
linear maximum likelihood regression was used to estimate the ECx values. Inhibitions lower
or equal to 0% or greater or equal to 100% were replaced by 0.10 and 99.9, respectively.
Fisher's Exact test (p≤0.05) was used to determine the threshold concentrations for daphnid
immobility.

The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH,
Naheweg 15, D-52477 Alsdorf) was used for these calculations.

The biological endpoints were expressed based on the nominal and on mean measured
concentrations.

Immobility :

Nominal and Measured Exposure Concentrations :

Validity criteria fulfilled:

yes

Conclusions:

When daphnids were exposed to Florhydral-Alcohol (GR-82-4337) under the test conditions of an OECD 202 study, the following end-points were derived :
48 hour EC50 =12.2 mg/L
48 hour EC0 = 4.70 mg/L
48 hour EC100 = 50.0 mg/L

Description of key information

AQUATIC TOXICITY :

FLORHYDRAL

Daphnia 48 hour EC50 = 2.26 mg/L

Additionally, full base-set aqautox studies are available for Florhydral-Acid (GR-81-6079) and Florhydral-Alcohol (GR-82-4332). Florhydral-Acid is the most relevant transformation product of FLORHYDRAL (via direct oxidation of the aldehyde group). The end-points determined are, as follows :

FLORHYDRAL-ACID (GR-81-6079) :

Daphnia 48 hour EC50 = 47.6 mg/L

FLORHYDRAL-ALCOHOL (GR-82-4332) :

Daphnia 48 hour EC50 = 12.2 mg/L

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

2.26 mg/L

Additional information

AQUATIC TOXICITY :

FLORHYDRAL

Daphnia 48 hour EC50 = 2.26 mg/L

Additionally, full base-set aqautox studies are available for Florhydral-Acid (GR-81-6079) and Florhydral-Alcohol (GR-82-4332). Florhydral-Acid is the most relevant transformation product of FLORHYDRAL (via direct oxidation of the aldehyde group). The end-points determined are, as follows :

FLORHYDRAL-ACID (GR-81-6079) :

Daphnia 48 hour EC50 = 47.6 mg/L

FLORHYDRAL-ALCOHOL (GR-82-4332) :

Daphnia 48 hour EC50 = 12.2 mg/L