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

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Final Report : 27 September 1991

First Test : 26 November 1990 to 03 December, 1990
Second Test : 16 April, 1991 to 22 April,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 203 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 203 (Fish, Acute Toxicity Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

Fish were exposed to five individual concentration levels during the 2nd main test for 96 hours, as well as a Blank Control and a Solvent Control.

The nominal test exposure concentrations were :
0.76 mg/L
1.37 mg/L
2.47 mg/L
4.44 mg/L
8.00 mg/L

Only samples of the 0.76 mg/L, 2.47 mg/L and 8.00 mg/L samples were analysed, and only at a maximum of 2 out of the 4 renewals. Solvent Control samples were also analysed : all detects in Solvent Control samples were below the LOQ of 0.2 mg/L.

Vehicle:

yes

Remarks:

The vehicle was Acetone and was employed at a maximum concentration of 0.1 mL/L in the exposure solutions.

Details on test solutions:

The nominal test exposure concentrations were :
0.76 mg/L
1.37 mg/L
2.47 mg/L
4.44 mg/L
8.00 mg/L

Exposure solutions prepared using an Acetone fortification of the test item. The maximum loading of Acetone in the exposure solutions was 0.1 mL/L.

From the limited analytics performed during the study for freshly prepared exposure solutions (0 hour) and Aged solutions (24 hours old - prior to renewal), the Nominal and Actual concentrations determined were, as follows :

Nominal 0.76 mg/L Actual Concn. 0.45 mg/L
Nominal 2.47 g/L Actual Concn. 1.45 mg/L
Nominal 8.0 mg/L Actual Concn. 4.69 mg/L

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

14.5 to 16.0 °C (Test 2)

pH:

pH 8.0 to 8.5 (Test 2)

Nominal and measured concentrations:

From the limited analytics performed during the study for freshly prepared exposure solutions (0 hour) and Aged solutions (24 hours old - prior to renewal), the Nominal and Actual concentrations determined were, as follows :

Nominal 0.76 mg/L Actual Concn. 0.45 mg/L
Nominal 2.47 g/L Actual Concn. 1.45 mg/L
Nominal 8.0 mg/L Actual Concn. 4.69 mg/L

No detects at or above LOQ corresponding to the Retention Time of the test item were observed in the Solvent Control samples.

High deviation between Nominal and measured starting concentrations measured by HPLC-UV/VIS were observed : Time 0 fresh exposure solutions observed to be between 51.9 and 96.5% of Nominal. Significant losses (presumably oxidised in to the Carboxylic Acid metabolite) were observed over the 24 hour renewal period.

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

1.082 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95%CI = 0.696 - 1.489 mg/L

Validity criteria fulfilled:

not specified

Conclusions:

The 96-hour LC50 of FLORHYDRAL to Rainbow Trout in this study was determined to be 1.082 mg/L, based on measured values.

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.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May, 2016 to 14 June, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

17 July, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Analytical monitoring:

yes

Details on sampling:

Samples were taken for analysis from each concentration level at
- The start of exposure;
- Before and After renewal;
- At the end of the Exposure.

Vehicle:

no

Details on test solutions:

Following a preliminary range-finding study, the following Nominal exposure concentrations were chosen for the definitive study :

Control
0.857 mg/L
1.54 mg/L
2.78 mg/L
5.00 mg/L
9.00 mg/L

Weighed test sample (0.0340 g) was added into Erlenmeyer flask filled with dilution water (3.4 L) to produce 10.0 mg/L as nominal concentration. Maximum, two stock solutions (3.4 L × 2) were prepared. 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 the test vessel to prepare the test solution, and then the airtight lid was set immediately. In addition, the stock solution was prepared every day.

Test organisms (species):

Oryzias latipes

Details on test organisms:

Test organisms :
Species Medaka (Oryzias latipes)
Reason for selection of species
Species recommended in the test guidelines
Supplier CERI Kurume (in-laboratory production)
Size Total length 2.3±1.2 cm,
Allocation Medaka was allocated at random to each test group.
Acclimation
Hatching date October 27, 2015 (age at the start of exposure; six-month-old)
Water Dechlorinated tap water
Type of acclimation
Flow-through regime
Dissolved oxygen concentration
At least 80% of air saturation value
Temperature 24±1°C
Lighting condition Room light, 16-hour light/8-hour dark
Feed Feed of Medaka fly for education (KYORIN)
Feeding amount and frequency
Amount corresponding to 3% of body weight was fed every day.
Use of medicament for external disinfection
None
Duration of acclimation
49 days (March 28, 2016 to May 16, 2016)
Mortality of test groups
0 % (during 7 days before the start of exposure)
Feed withdrawal 24 hours before the start of exposure

Test type:

semi-static

Water media type:

freshwater

Remarks:

Dilution water :Dechlorinated tap water, aerated sufficiently and temperature-controlled, was used. The result of chemical characteristics of the dilution water filled the criteria that provided with the standard operating procedure of this laboratory.

Limit test:

no

Total exposure duration:

96 h

Hardness:

37 mg/L as CaCO3

Test temperature:

23.2 to 23.9 °C

pH:

pH 7.3 - 7.9

Dissolved oxygen:

8.8 to 8.3 mg O2/L

Conductivity:

15 mS/m

Nominal and measured concentrations:

Analysis of Parent:
All Fresh exposure solutions were in the range of 91.9 to 101% of the Nominal.
After 24 hours, the Aged exposure solutions demonstrated a significant decrease in Parent to between 33.6 to 80.7% of Nominal concentrations

Quantification of Metabolites - Florhydral Acid and Florhydral Alcohol :
Florhydral-Acid and Florhydral-Alcohol were determined in all of the water samples.
Only detects of the Florhydral-Acid was made in the Fresh exposure solution at 0 and 24 hours constituting 1.05 and 0.93% of the nominal dose at the 9.76 mg/L Nominal exposure concentration.
No Florhydral-Alcohol was detected in any of the Fresh starting exposure solutions.

After 24 hours of ageing, between 4.50 and 24.7% of the applied Nominal dose was present as Florhydral-Acid.
After 24 hours of ageing, between 4.37 and 24.6% of the applied Nominal dose was present as Florhydral-Alcohol.

Adding Parent + Florhydral-Acid + Florhydral-Alcohol together, the Mass Balance of 24 hour old exposure solutions was found to be between 77.0 and 94.1% of the applied dose.

Details on test conditions:

Test apparatus and equipment :
Test vessel 3 L Glass tank (diameter: 16 cm, depth: 17 cm)
Cover on test vessel Airtight transparent plastic lid
Water bath Plastic tank
Warming/cooling unit; Type HCA 250 (Sato craft)

Test conditions :
Type of test Semi-static (renewal every 24 hours), closed system with limited
headspace
Exposure duration 96 hours
Test concentration 9.00, 5.00, 2.78, 1.54, 0.857 mg/L as nominal concentration (a geometric series with a factor of 1.8)
The test concentrations and the geometric factor were decided from the results of preliminary study.
The results of the preliminary study are shown in Additional data.
Control Dilution water without the test item.
Replicate 1 replicate/test level
Number of organisms 7 fish/test level
Volume of test solution Approx. 2.7 L/test level
Temperature of test solution
24±1°C
Aeration No aeration
pH adjustment No adjustment
Lighting condition Room light, 16-hour light/8-hour dark
Feeding No feeding

Reference substance (positive control):

yes

Remarks:

Reference substance Copper (II) sulfate pentahydrate (JIS special grade, Wako Pure Chemical Industries, Lot No. CTP2097) Test period May 23 to May 27, 2016 96-hour LC50 0.25 mg/L

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

4.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.29 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Parent + Florhydral-Acid + Florhydral Alcohol

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

1.89 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

2.44 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Parent + Florhydral-Acid + Florhydral-Alcohol

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

7.82 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

8.51 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

other: Parent + Florhydral-Acid + Florhydral-Alcohol

Basis for effect:

mortality (fish)

Results with reference substance (positive control):

Acute toxicity test of a reference substance is periodically conducted. The latest data is shown below.
Reference substance Copper (II) sulfate pentahydrate (JIS special grade, Wako Pure Chemical Industries, Lot No. CTP2097)
Test period May 23 to May 27, 2016
96-hour LC50 0.25 mg/L
This value was within the stipulated range (mean ± 2S.D.) [mean ± S.D.: 0.53 ± 0.19 mg/L (n=58)] to background data in this laboratory. All of the values shown above for the reference substance were converted into Copper (II) sulfate value.

Sublethal observations / clinical signs:

Cumulative Mortality :

Validity criteria fulfilled:

yes

Conclusions:

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, 96-hour LC50 was 4.60 mg/L, based on geometric mean measured concentrations of parent (test item). The definitive study was conducted by semi-static regime (renewal every 24 hours) and closed system with headspace to maintain the test item concentration in consideration of the volatility, oxidizability and deoxidizability of the test item. The maintenance rate of test item concentrations in the test solution during exposure was 36.1-79.9%. It was confirmed that the measured concentrations of both converted products (GR-81-6079 and GR-82-4337) during exposure were increased with decrease the measured concentration of the test item sequentially.

The 96 hour LC50 based on the geometric mean measured concentration of parent + metabolites (converted products) was 5.29 mg/L.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

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 203 (Fish, Acute Toxicity Test)

Version / remarks:

July, 1992

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%
Expiry Date : 22 July, 2015

Analytical monitoring:

yes

Details on sampling:

GR-81-6079 was determined to be stable in a preliminary range-finding study. A static exposure study was, therefore, performed.

Samples were taken at the ebginning of the exposure (0 hour) and at the end of the exposure (96 hours) for analysis.

Vehicle:

no

Details on test solutions:

Reconstituted water according to OECD guideline No. 203 was used to dilute the test item
and to keep the fish before and during the period of the test.
Local tap water was treated by reverse osmosis and ion-exchanger to prepare deionised
water. Therefore a contamination with heavy metals, pesticides and TOC (Total Organic
Carbon) is excluded.
The reconstituted water consists of deionised water and salts in the following final
concentrations:
A) CaCl2 * 2 H2O 294.0 mg/L
B) MgSO4 * 7 H2O 123.0 mg/L
C) NaHCO3 64.8 mg/L
D) KCl 5.75 mg/L
The required amount of reconstituted water was prepared within one month before use.
During storage, reconstituted water was aerated. After aeration of several hours, the
physico-chemical characteristics of the reconstituted water were determined according to
the requirements of the test guideline.

Preparation of Test Solutions :
Reconstituted water was used for the controI.
Stock solutions were prepared once, one day before start of the exposure period. At day of
preparation, the test item was separately weighed into 5 mL snap-on glass flasks. The glass
vessels were stored (30 minutes) with closed lids until preparation of each stock solution. Four
separate stock solutions were prepared. The 5 mL snap-on glass flasks were
rinsed with test medium and the test item were transferred in 5 and 10 L measuring flasks.
The 5 mL snap-on glass flasks were visually checked on remaining test item. Thereafter, the
measuring flasks were filled with test medium up to the calibration mark. The final sock
solution were stirred for one day at room temperature in dark.
At day of exposure start, the stock solutions were visually observed for undissolved test item
particles, after 30 minutes without stirring. In all stock test item particles were visible in the
medium and on the surface of the stock solution. Therefore, the stock solutions were filtered
through a glass-fibre filter with an average retention capacity of 0.6 μm (Type: MN 85/70 BF,
diameter: 110 mm). The filtrates were visually checked using Tyndall effect, and no particles
were visible. Before the stock solutions were used to prepare the test solutions the stock
solutions were combined in a 200 L stainless steel tank. This combined stock solution was
used to prepare the test solution used for exposure.
The volumes of the stock solutions were large enough to prepare the test solutions and all
analytical samples.

The test solutions were prepared by weighing the corresponding amounts of stock and dilution
medium in the test vessels.

Test concentrations (nominal): 4.3, 9.4, 20.7, 45.5, 100% of a filtrate at a
loading rate of 100 mg/L plus control.

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Characterisation of the Test System (Test Organism) :
 Fish Species: freshwater fish Oncorhynchus mykiss (WALBAUM, 1792; rainbow trout)
 Supplier: Forellenzucht Trostadt GbR
 Hatching date: ca. November 20, 2014
 Date of arrival: January 22, 2015
 Average body weight of fish: 0.692 g/fish (assessed from the total weight of a sample
consisting of 10 fishes);
 Average standard length of fish: 4.87 cm

Holding Conditions :
 Material of stock vessel: glass fibre reinforced plastic
 Amount of water per stock vessel: 350 L
 Depth of water in the stock vessel: 55 cm
 Estimated number of fish kept as a stock per holding vessel (batch size): 83
 Fish loading: 0.144 g/L
 Water: reconstituted water + 0.1% of reconstituted seawater, see section 13.4
 Renewal of water: once or twice per week
 Temperature of water: 15±2 C; min/max 14.6 °C/14.8° C (during 14 days before start of
exposure)
 Food: commercial rainbow trout food (Aller Performa Gr.1, Aller Aqua, Denmark)
 Amount of food: approximately 2% of fish fresh weight per day
 Feeding frequency: daily ration fed in two portions. No feeding 20.5 hours before start of
exposure, see section 18.
 Photoperiod: 12 h light / 12 h dark
 Light intensity: 100–1000 lx
 Adaptation period (>12 d): 43 days
 Mortalities during the period of 7 d before start of the test (<5%): 4.8%.

Test type:

static

Water media type:

freshwater

Remarks:

Test Medium : Reconstituted water according to OECD guideline No. 203 was used to dilute the test item and to keep the fish before and during the period of the test.

Limit test:

no

Total exposure duration:

96 h

Hardness:

246.5 mg/L as CaCO3

Test temperature:

14.87 °C (Range : 13.5 - 15.1 °C)

pH:

pH 6.3 to 7.6

Conductivity:

663 microS/cm2

Nominal and measured concentrations:

The Nominal exposure concentrations were expressed as a %of filtrate prepared from a 100 mg/L starting solution :

Nominal Measured Concn. (mg/L)
(% filtrate of 100 mg/L) 0 hours 96 hours

100% 79.6 66.2
45.5% 35.4 30.9
20.7% 17.7 23.3
9.4% 6.97 7.44
4.3% 3.26 2.60

The concentrations of GR-81-6079 measured in the samples of the test solution taken on
days 0 and 4 days, ranged from 60–113% of the nominal loading rate. The measured
concentrations at start of the test ranged between 74–86% and at the end between 60–
113% of the nominal loading rate. The concentrations in the test solutions were stable
throughout the exposure period (range: 80–132% of initial measured concentrations).

Details on test conditions:

Exposure Conditions :
– Volume of test solution per test
vessel:17 L
– Depth of test solution in the test
vessels:approx. 21 cm
– Number of fish per test vessel: 7
– Length of fish measured before start of exposure (range):
4.5–5.5 cm (assessed from the total weight of a
sample consisting of 10 fishes); determined on a
representative group of 10 fish on March 03, 2015);
– Average body weight of fish: 0.692 g/fish (assessed from the total weight of a
sample consisting of 10 fishes); determined on a
representative group of 10 fish on March 03, 2015);
– Fish loading: 0.285 g per L
– Renewal of test solutions during exposure:None
– Feeding: None
– Photoperiod: Light/dark - 12 h/12 h
– Light intensity: 427–565 lx;
– Temperature: 15.0–15.5 °C (n = 22; manual measurement in the
test vessels); 13.5–15.1 °C (n = 196; automatic measurement in
the control test vessel);
– Aeration: Yes (permanent)

Procedures :
Food was withheld from the fish for 20.5 hours prior to the start of exposure.
 The test vessels were placed in a temperature controlled water bath at a temperature
set to 15°C.
 The fish were added to the test vessels after sequentially adding one fish to the preselection
vessel designated to a specific treatment level (1 L Bellaplast containers
(Polystyrol)) until each pre-selection vessel contained the desired number of fish.
Thereafter, the fish of each pre-selection vessel were transferred to the designated test
vessel.
 All fish in the test were observed 3 hours, 6 hours, and 24, 48, 73 and 96 hours after
start of exposure. Any dead fish were recorded and removed from the test vessels. Fish
were considered dead if there was no visible movement (e.g. gill movement) and if
touching of the caudal peduncle produced no reaction.
 All fish in the test were observed daily for other than lethal effects: effects on the
appearance, size and behaviour of the fish that make them clearly distinguishable from
the control animals, e.g. different swimming behaviour, different reaction to external
stimuli, changes in appearance of the fish were recorded.
 Aeration was controlled daily in all test vessels on a visual basis; since aeration was fine
at each inspection, no adjustment was performed.
 After the end of the exposure period the fish were anaesthetised with buffered MS222
(3-aminobenzoic acid ethyl ester methanesulfonate salt, Tricain) and killed thereafter,
and disposed according to currently applicable local regulations.
 After sampling for chemical analysis, any unused test solutions were treated with
activated carbon and disposed in agreement with currently applicable local legislation.

Water Quality Measurements :
After temperature adaptation of the test solutions, and daily during the test, the following
parameters were measured and recorded for each test concentration and the control in
each test vessel:
- temperature,
- pH,
- dissolved oxygen content in % of air saturation value (ASV) and in mg/L,
Temperature was additionally recorded once per hour throughout the test in one test vessel.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

20.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

25.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

Therefore, it can be concluded that the LC50 (96 h) to Oncorhynchus mykiss (Rainbow
Trout) based on nominal concentrations is 25.4% of a filtrate at a loading rate of 100 mg/L
and 20.8 mg test item/L based on measured concentrations.
The NOEC (96 h) based on statistical evaluation of mortality is 20.7% of a filtrate at a
loading rate of 100 mg test item/L and 20.3 mg test item/L based on measured
concentrations.
The highest concentration showing no considerable adverse effects on the behaviour or
appearance of the fish is 4.3% of a filtrate at a loading rate of 100 mg test item/L, which is
2.91 mg test item/L based on the time-weighted mean measured concentrations.

Sublethal observations / clinical signs:

Cumulative mortality of the fish :

LC50 values of the parameter "mortality of fish" based on statistical evaluation of

biological results and nominal concentrations in % of a filtrate at a loading rate of 100 mg/L

and measured concentrations in mg test item/L :

 :

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 of GR-81-6079 to O. mykiss was :
20.8 mg/L (measured)
25.4 mg/L (Nominal)

A clear concentration-response relationship was observed.

The concentrations of GR-81-6079 measured in the samples of the test solution taken on
days 0 and 4 days, ranged from 60–113% of the nominal loading rate. The measured
concentrations at start of the test range between 74–86% and at the end between 60–113%
of the nominal loading rate. The concentrations in the test solutions were stable throughout
the exposure period (range: 80–132% of initial measured concentrations).

The biological endpoints were expressed based on nominal concentrations. In addition,
since all concentration level were measured the biological endpoints were expressed based
on the geometric mean concentration calculated for each concentration level.
Therefore, it can be concluded that the LC50 (96 h) to Oncorhynchus mykiss (Rainbow
Trout) based on nominal concentrations is 25.4% of a filtrate at a loading rate of 100 mg/L
and 20.8 mg test item/L based on measured concentrations.

The NOEC (96 h) based on statistical evaluation of mortality is 20.7% of a filtrate at a
loading rate of 100 mg test item/L and based on measured concentrations 20.3 mg test
item/L.

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 March, 2015 - 23 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 203 (Fish, Acute Toxicity Test)

Version / remarks:

July,1992

Deviations:

no

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:

Water samples were taken and analysed for all exposure levels and the Control on :
0 hours - Fresh
48 hours - Aged
48 hours - Fresh, Renewal
96 hours - Aged

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: 2.0) were tested in the definitive test:
0.32, 0.63, 1.25, 2.50, 5.00 and 10.0 mg test item/L.
Additionally, fish were exposed under control conditions (non-treated test medium).
One test replicate vessel was used for each treatment, and one for the control. Each vessel
contained seven fish.

Preparation of Test Solutions :
Reconstituted water was used for the control.
Stock solutions were prepared twice, on day 0 and 2 of the exposure period. At each day of
preparation, two batches of stock solutions were prepared. One was used to prepare test
solutions used for conditioning of the test vessels (stock solution a0) and a2)) and the second
was used to prepare the test solution used for exposure (stock solution b0) and b2)).
At day 0 the stock solutions were prepared by mixing 0.4000 and 0.4006 g of the test item in
40 L of test medium (nominal concentration 10 mg test item/L).
Stock solution a0): The test item was weighed into a Teflon weighing boat. The total amount of
test item was transferred into a 10 L measuring flask and filled up with test medium. This
solution was stirred for one hour on a magnetic stirrer at room temperature in dark. Thereafter,
the solution was transferred in a 200-L stainless steel tank and 30 L of test medium was added
using a 10 L measuring flask, and the solution was stirred for further half an hour with a
magnetic stirrer at room temperature in dark. Thereafter, the stock solution was used to
prepare the test solutions (conditioning of stock solution b0) beaker and test vessels).
Stock solution b0): The test item was weighed into a Teflon weighing boat. The total amount of
test item was transferred into a pre-conditioned 54 L glass-balloon bottle (the glass-ballon
bottle was conditioned/rinsed with stock solution a0) shortly before used). 40 L of test medium
was added to the bottle using a 10 L measuring flask. This solution was stirred for 75 minutes
on a magnetic stirrer at room temperature in dark. Thereafter, the stock solution was used to
prepare the test solutions (exposure period).
At day 2 the stock solutions were prepared by mixing 0.2005 and 0.2009 g of the test item in
20 L of test medium (nominal concentration 10 mg test item/L).
Stock solution a0): 20 L of test medium was added to a 200 L stainless steel tank using a 10 L
measuring flask. The test item was weighed into a Teflon weighing boat and the total weighing
boot was transferred to the 200 L stainless steel tank. This solution was stirred for 75 minutes
on a magnetic stirrer at room temperature in dark. Thereafter, the stock solution was used to
prepare the test solutions (conditioning of Stock solution b0) beaker and test vessels).
Stock solution b0): 20 L of test medium was added to a 54 L glass-balloon bottle using a 10 L
measuring flask. The test item was weighed into a Teflon weighing boat and the total weighing
boot was transferred to the 54 L glass-balloon bottle. This solution was stirred for one hour on
a magnetic stirrer at room temperature in dark. Thereafter, the stock solution was used to
prepare the test solutions (exposure period).

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

Characterisation of the Test System (Test Organism) :
 Fish Species: freshwater fish Oncorhynchus mykiss (WALBAUM, 1792; rainbow trout)
 Supplier: Forellenzucht Trostadt GbR
 Hatching date: ca. November 20, 2014
 Date of arrival: January 22, 2015
 Average body weight of fish: 0.692 g/fish (assessed from the total weight of a sample
consisting of 10 fishes); 
Average standard length of fish: 4.87 cm (assessed in a sample consisting of 10 fish);
determined on a representative group of 10 fish on March 03, 2015).

Holding Conditions :
 Material of stock vessel: glass fibre reinforced plastic
 Amount of water per stock vessel: 350 L
 Depth of water in the stock vessel: 55 cm
 Estimated number of fish kept as a stock per holding vessel (batch size): 175
 Fish loading: 0.346 g per L
 Water: reconstituted water + 0.1% of reconstituted seawater, 
Renewal of water: once per week
 Temperature of water: 15±2 °C; min/max 14.7 °C/14.8 C (during 7 days before start of
exposure)
 Food: commercial rainbow trout food (Aller Performa Gr.1, Aller Aqua, Denmark)
 Amount of food: approximately 2% of fish fresh weight per day
 Feeding frequency: daily ration fed in two portions on workdays. No feeding on the two
days before start of exposure.
 Photoperiod: 12 h light / 12 h dark
 Light intensity: 100–1000 lx
 Adaptation period (>12 d): 46 days
 Mortalities during the period of 7 d before start of the test (<5%): 1.7%

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted water according to OECD guideline No. 203 was used to dilute the test item and to keep the fish before and during the period of the test.

Limit test:

no

Total exposure duration:

96 h

Hardness:

257.2 mg/L as CaCO3

Test temperature:

14.6 - 16.2 °C

pH:

pH 7.5 - 7.8

Dissolved oxygen:

8.6 - 9.3 mg O2/L

Conductivity:

692 microS/cm2

Nominal and measured concentrations:

The concentrations of GR-82-4337 measured in the samples of the test solution taken on
days 0, 2 and 4 days, ranged from 51–94% of the nominal concentrations. The measured
concentrations in freshly prepared test solution ranged between 55–80% at start of the test
and 77–94% at first renewal, and in aged test solution at end of first renewal period between
51–65% and at the end of the second renewal period between 76–88% (end of the test) of
the nominal concentrations. The concentrations in the test solutions were stable within each
renewal period of 2 days (range: 82–103% of initial measured concentrations).

Details on test conditions:

Exposure Conditions :
– Volume of test solution per test vessel:18 L
– Depth of test solution in the test vessels: Approx. 21 cm
– Number of fish per test vessel: 7
– Length of fish measured before start of exposure (range):
4.5–5.5 cm; determined on a representative group of
10 fish on March 03, 2015);
– Average body weight of fish: 0.692 g/fish; determined on a representative group
of 10 fish on March 03, 2015);
– Fish loading: 0.285 g per L
– Renewal of test solutions during exposure: One renewal after 48 hours
– Feeding: None
– Photoperiod: Light/dark - 12 h/12 h
– Light intensity: 501–628 lx;
– Temperature: 14.6–16.2 °C (n = 39; manual measurement in the test vessels);
14.3–15.2 °C (n = 195; automatic measurement in the control test vessel);
– Aeration: Yes (permanent)

Procedures :
 Food was withheld from the fish for two days prior to the start of exposure.
 The test vessels were placed in a temperature controlled water bath at a temperature
set to 15 °C.
 The fish were added to the test vessels after sequentially adding one fish to the preselection
vessel designated to a specific treatment level (1 L Bellaplast containers
(Polystyrol)) until each pre-selection vessel contained the desired number of fish.
Thereafter, the fish of each pre-selection vessel were transferred to the designated test
vessel.
 All fish in the test were observed 3 hours, 6 hours, and 24, 48, 52, 72 and 96 hours after
start of exposure. Any dead fish were recorded and removed from the test vessels. Fish
were considered dead if there was no visible movement (e.g. gill movement) and if
touching of the caudal peduncle produced no reaction.
 All fish in the test were observed daily for other than lethal effects: effects on the
appearance, size and behaviour of the fish that make them clearly distinguishable from
the control animals, e.g. different swimming behaviour, different reaction to external
stimuli, changes in appearance of the fish were recorded.
 Aeration was controlled daily in all test vessels on a visual basis; since aeration was fine
at each inspection, no adjustment was performed.
 After the end of the exposure period the fish were anaesthetised with buffered MS222
(3-aminobenzoic acid ethyl ester methanesulfonate salt, Tricain) and killed thereafter,
and disposed according to currently applicable local regulations.
 After sampling for chemical analysis, any unused test solutions were treated with
activated carbon and disposed in agreement with currently applicable local legislation.

Water Quality Measurements :
After temperature adaptation of the test solutions, and daily during the test, the following
parameters were measured and recorded for each test concentration and the control in
each test vessel:
- temperature,
- pH,
- dissolved oxygen content in % of air saturation value (ASV) and in mg/L,
At the day the test solutions were renewed during the exposure period, the physicalchemical
parameter (temperature, pH, dissolved oxygen) were measured at the day of
preparation in the aged and freshly prepared test solutions after temperature adaptation.
Temperature was additionally recorded twice per hour throughout the test in one test vessel.

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.81 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

8.04 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

A clear concentration-response relationship was observed.

Mortality was observed in the highest test concentration C6 (10.0 mg test item/L). No
mortality was observed in the control and the test item treated group (C2, C4 and C5, 0.63,
2.5 and 5.0 mg test item/L) during the 96-hour exposure period. One fish died at
concentration levels C1 and C3 (0.32 and 1.25 mg test item/L). There is no clear indication
of these mortalities being substance-related given that no mortalities occurred during the 96
hour exposure period in the two exposure concentrations above C3 (1.25 mg test item/L).

It can be concluded that the LC50 (96 h) to Oncorhynchus mykiss (Rainbow
Trout) is 8.04 mg test item/L based on nominal concentrations and 5.81 mg test item/L
based on measured concentrations.

The NOEC (96 h) based on statistical evaluation of mortality is 5.00 mg test item/L based on
nominal concentrations and 3.38 mg test item/L based on the time-weighted mean
concentration.

Sublethal observations / clinical signs:

Cumulative Mortality of Fish :

Validity criteria fulfilled:

yes

Conclusions:

The 96 hour LC50 of GR-82-4337 to O. mykiss was :
5.81 mg/L (measured)
8.04 mg/L (Nominal)

The concentrations of GR-82-4337 measured in the samples of the test solution taken on
days 0, 2 and 4 days, ranged from 51–94% of the nominal concentrations. The measured
concentrations in freshly prepared test solution ranged between 55–80% at start of the test
and 77–94% at first renewal, and in aged test solution at end of first renewal period between
51–65% and at the end of the second renewal period between 76–88% (end of the test) of
the nominal concentrations. The concentrations in the test solutions were stable within each
renewal period of 2 days (range: 82–103% of initial measured concentrations).

The biological endpoints were expressed based on nominal concentrations. In addition,
since all concentration levels were measured, the biological endpoints were expressed
based on the time-weighted-mean concentration calculated for each concentration level.
Therefore, it can be concluded that the LC50 (96 h) to Oncorhynchus mykiss (Rainbow
Trout) based on nominal concentrations is 8.04 mg test item/L and based on measured
concentrations, 5.81 mg test item/L.

The NOEC (96 h) based on statistical evaluation of mortality is 5.00 mg test item/L based on
nominal concentrations and 3.38 mg test item/L based on the time-weighted mean
measured concentration.

Description of key information

AQUATIC TOXICITY :

FLORHYDRAL

Fish 96 hour LC50 = 4.6 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) :

Fish 96 hour LC50 = 20.8 mg/L

FLORHYDRAL-ALCOHOL(GR-82-4332) :

Fish 96 hour LC50 = 5.81 mg/L

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.6 mg/L

Additional information

AQUATIC TOXICITY :

FLORHYDRAL

Fish 96 hour LC50 = 4.6 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) :

Fish 96 hour LC50 = 20.8 mg/L

FLORHYDRAL-ALCOHOL(GR-82-4332) :

Fish 96 hour LC50 = 5.81 mg/L